Disruption of Cell-Cell Adhesion Codes
Underlies the Unique X-linked
Inheritance Pattern of Protocadherin 19
Girls Clustering Epilepsy
Daniel Tyler Pederick
B.Sc. (Biomedical Science), Honours (Biochemistry)
A thesis submitted in fulfilment of the requirements for the degree of Doctor
of Philosophy
Department of Molecular and Cellular Biology
School of Biological Science
The University of Adelaide, Australia
February 2017
i
Contents
Abstract ................................................................................................................................. iii
PhD Thesis Declaration ......................................................................................................... v
Publications ......................................................................................................................... viii
Conference Oral Presentations .............................................................................................. ix
Awards .................................................................................................................................. xi
Introduction .......................................................................................................................... 12
1.1 Epilepsy ...................................................................................................................... 13
1.2 Protocadherin 19 Girls Clustering Epilepsy .............................................................. 16
1.3 Pcdh19 expression in the mammalian brain .............................................................. 20
1.4 The Cadherin Superfamily ......................................................................................... 22
1.5 Adhesive Functions of Protocadherins in the Mammalian Brain .............................. 27
1.6 Neural Function of PCDH19 ...................................................................................... 33
1.7 What causes the unusual inheritance of PCDH19-GCE? .......................................... 35
1.8 Pcdh19 Null Mouse.................................................................................................... 37
1.9 Project Rationale ........................................................................................................ 38
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is
Dispensable for Brain Development in Mice ...................................................................... 39
2.1 Summary .................................................................................................................... 40
CHAPTER 3: Abnormal cell sorting is associated with the unique X-linked inheritance of
PCDH19 epilepsy ................................................................................................................ 58
3.1 Summary .................................................................................................................... 59
CHAPTER 4: General Discussion ..................................................................................... 124
ii
CHAPTER 5: Future Directions ........................................................................................ 134
5.1 Investigating Connectivity between PCDH19 WT and PCDH19 null neurons ....... 136
5.2 Is Interneuron Distribution Affected by Mosaic Pcdh19 Expression?..................... 138
5.3 Can Epileptiform Brain Activity be Rescued after Abnormal Cell Sorting has
Occurred? ....................................................................................................................... 143
5.4 Investigating the Combinatorial Activity of other NC PCDHs In Vivo ................... 144
5.5 Concluding remarks ................................................................................................. 146
Additional Methods............................................................................................................ 147
References .......................................................................................................................... 148
iii
Abstract
Epilepsy is a disease of the central nervous system (CNS) caused by increased neuronal
activity resulting in seizures and often loss of consciousness. Epilepsy can result from both
traumas to the brain or a genetic predisposition. Recent advances in DNA sequencing
technology has identified many forms of epilepsy caused by mutations in single genes.
Although such monogenic epilepsies are rare, investigating their underlying molecular
mechanisms provides important insights into pathways and processes that cause seizures and
assists in the application of pharmacotherapy and surgical strategies.
The second most common form of monogenic epilepsy is Protocadherin 19 Girls Clustering
Epilepsy (PCDH19-GCE) caused by mutation of the X-linked gene PCDH19. This disorder
is characterised by clusters of febrile seizures beginning in early childhood that is often
accompanied by variable intellectual disability and autism spectrum disorder. The most
striking feature of PCDH19-GCE is its unique X-linked inheritance pattern; heterozygous
females with PCDH19 mutations are affected whereas hemizygous males are not. It is
hypothesised that the mixture of PCDH19-WT and PCDH19-mutant neurons (generated by
random X-inactivation in female brains) causes abnormal neuronal connections leading to
disease. However, there is no experimental evidence supporting this hypothesis and the
cellular and molecular mechanisms underpinning this unique inheritance pattern are
unknown.
To better understand how mutation of PCDH19 leads to the unique X-linked inheritance
pattern this thesis uses a Pcdh19 null mouse, cell culture assays and unique CRISPR-Cas9
engineered mouse models. It is shown that heterozygous and homozygous deletion of
Pcdh19 in mice does not cause any gross brain morphological abnormalities and that Pcdh19
iv
null neurons are present within the correct layers in the cortex despite their slight increase in
migration potential in vitro.
Using cultured K562 cells it is shown that PCDH19 and other non-clustered (NC) PCDH
members contribute to combinatorial adhesion codes that dictate specific cell-cell
interactions. Similarly, mosaic expression of Pcdh19 in heterozygous mice leads to abnormal
cell sorting in the developing cortex such that cells separate into PCDH19 positive and
negative patches, correlating with altered brain network activity consistent with changes that
can underlie seizures in adult mice. Deletion of Pcdh19 in heterozygous embryos using
CRISPR-Cas9 technology eliminates this incompatibility of adhesion codes and prevents
abnormal cell sorting from occurring. In addition, variable cortical folding malformations in
PCDH19-GCE epilepsy patients were identified. Collectively these results highlight the role
of PCDH19 in determining specific adhesion codes during brain development and how
disruption of these codes is associated with the unique X-linked inheritance pattern of
PCDH19-GCE. Importantly, a framework is provided for investigating how this abnormal
neuronal segregation phenotype leads to seizures and cognitive deficits.
v
PhD Thesis Declaration
I certify that this work contains no material which has been accepted for the award of any
other degree or diploma in my name in any university or other tertiary institution and, to the
best of my knowledge and belief, contains no material previously published or written by
another person, except where due reference has been made in the text. In addition, I certify
that no part of this work will, in the future, be used in a submission in my name for any other
degree or diploma in any university or other tertiary institution without the prior approval of
the University of Adelaide and where applicable, any partner institution responsible for the
joint award of this degree.
I give consent to this copy of my thesis when deposited in the University Library, being
made available for loan and photocopying, subject to the provisions of the Copyright Act
1968.
The author acknowledges that copyright of published works contained within this thesis
resides with the copyright holder(s) of those works.
I also give permission for the digital version of my thesis to be made available on the web,
via the University's digital research repository, the Library Search and also through web
search engines, unless permission has been granted by the University to restrict access for a
period of time.
I acknowledge the support I have received for my research through the provision of an
Australian Government Research Training Program Scholarship.
Daniel Pederick
vi
Acknowledgements
I would firstly like to thank all my colleagues, friends and family for their support, kindness
and tolerance that has made my PhD adventure so enjoyable and rewarding.
I owe many of my achievements (and hopefully future success) to Prof. Paul Thomas who
has undoubtedly changed the way I approach my research. I appreciate that your door is
rarely ever closed, allowing me to discuss new experiments I have planned, even sometimes
letting me do them. This to me is priceless as it has helped me gain some key skills needed
to become an independent scientist. I am equally as grateful for all your support of my future
endeavours; hopefully we can form successful collaborations in the future.
Dr. James Hughes, you have been an excellent co-supervisor and friend over the years. I
thank you for your always insightful scientific and non-scientific discussions and also your
technical guidance in the lab. Without your assistance I doubt I would’ve been able to work
independently within the lab.
Prof. Jozef Gecz, thank you for your role as co-supervisor. Your “external” point of view is
always greatly valued and appreciated, as are the heated table tennis matches over the years
at past Epilepsy Retreats. Thanks also to members of your group who have assisted me
throughout my research.
I also appreciate all the help from all past/current lab members and collaborators specifically
Dale McAninch, Bryan Haines, Sandie Piltz and Kay Richards
I would also like to thank the all of the professional staff within the School of Biological
Sciences, especially Alan McLennan who has always been willing to help above and beyond
what his job requires.
vii
Staff at university provided services such as Adelaide Microscopy and Laboratory Animal
Services; I thank you for your time and efforts, especially Agatha Labrinidis and Tiffany
Boehm.
Thank you to all members of the Biochemistry/Molecular and Cellular Biology Disciplines
for providing such an excellent research environment and always being prepared to help.
I must also acknowledge my parents Greg and Mary and all of my relatives. Without your
support and belief it would not have been possible. Thank you also for allowing me to follow
my interest in science.
Finally, I thank my partner Gabby for her love and support over the past four years. If you
were not so understanding I doubt I would have been able to complete my PhD and maintain
my sanity. I look forward to more of your unwavering support in the future.
viii
Publications
Pederick, D.T., Homan, C.C., Jaehne, E.J., Piltz, S.G., Haines, B.P., Baune, B.T., Jolly, L.A.,
Hughes, J.N., Gecz, J., and Thomas, P.Q. (2016). Pcdh19 Loss-of-Function Increases
Neuronal Migration In Vitro but is Dispensable for Brain Development in Mice. Sci. Rep.
6, 26765.
*Submitted manuscript
Pederick, D.T., Richards, K.L., Piltz, S.G., Mandelstam, S.A., Dale, R.C., Scehffer, I.E.,
Gecz, J., Petrou, S., Hughes, J.N., Thomas, P.Q. (2017) Abnormal cell sorting is associated
with the unique X-linked inheritance of PCDH19 epilepsy. Submitted to: Science on
27.1.2017
ix
Conference Oral Presentations
Mosaic Expression of Pcdh19 Causes Segregation of PCDH19-wild type and PCDH19-null
Neurons in the Developing Mouse Cortex Leading to an Epileptic Phenotype
Daniel T. Pederick, Kay L. Richards, Sandra G. Piltz, Simone A. Mandelstam, Ian Dodd,
Ingrid E. Scheffer, Jozef Gecz, Steven Petrou, James N. Hughes and Paul Q. Thomas
Australian Society for Medical Research, South Australian Scientific Meeting (June 2016)
Mosaic Expression of Pcdh19 Causes Segregation of PCDH19-wild type and PCDH19-null
Neurons in the Developing Mouse Cortex Leading to an Epileptic Phenotype
Daniel T. Pederick, Kay L. Richards, Sandra G. Piltz, Simone A. Mandelstam, Ian Dodd,
Ingrid E. Scheffer, Jozef Gecz, Steven Petrou, James N. Hughes and Paul Q. Thomas
Adelaide Protein Group Student Awards (June 2016)
Mosaic Expression of Pcdh19 Causes Segregation of PCDH19-wild type and PCDH19-null
Neurons in the Developing Mouse Cortex Leading to an Epileptic Phenotype
Daniel T. Pederick, Kay L. Richards, Sandra G. Piltz, Simone A. Mandelstam, Ian Dodd,
Ingrid E. Scheffer, Jozef Gecz, Steven Petrou, James N. Hughes and Paul Q. Thomas
Gordon Research Seminar-Neurodevelopment (July 2016)
Mosaic Expression of Pcdh19 Causes Segregation of PCDH19-wild type and PCDH19-null
Neurons in the Developing Mouse Cortex Leading to an Epileptic Phenotype
Daniel T. Pederick, Kay L. Richards, Sandra G. Piltz, Simone A. Mandelstam, Ian Dodd,
Ingrid E. Scheffer, Jozef Gecz, Steven Petrou, James N. Hughes and Paul Q. Thomas
Gordon Research Conference-Neurodevelopment (August 2016)
x
Playing TAG with PCDH19: Investigating the Cellular and Molecular Mechanism of
PCDH19 Human Epilepsy
Daniel T. Pederick, Kay L. Richards, Sandra G. Piltz, Simone A. Mandelstam, Ian Dodd,
Ingrid E. Scheffer, Jozef Gecz, Steven Petrou, James N. Hughes and Paul Q. Thomas
ComBio 2016 (October 2016)
Mosaic loss of Pcdh19 in mice leads to abnormal cell sorting and epileptiform brain activity
Daniel T. Pederick, Kay L. Richards, Sandra G. Piltz, Simone A. Mandelstam, Ian Dodd,
Ingrid E. Scheffer, Jozef Gecz, Steven Petrou, James N. Hughes and Paul Q. Thomas
2016 ANZSCDB Adelaide Meeting
Mosaic loss of Pcdh19 in mice leads to abnormal cell sorting and epileptiform brain activity
Daniel T. Pederick, Kay L. Richards, Sandra G. Piltz, Simone A. Mandelstam, Ian Dodd,
Ingrid E. Scheffer, Jozef Gecz, Steven Petrou, James N. Hughes and Paul Q. Thomas
School of Biological Sciences Research Day, The University of Adelaide
xi
Awards
Best student poster - Robinson Research Institute Symposium (2014)
Robinson Research Institute Prize (Best presentation in the field of Reproduction, Pregnancy
or Child Health) - Australian Society of Medical Research South Australian Scientific
Meeting (2016)
Most outstanding oral presentation - Adelaide Protein Group Student Awards (2016)
Robinson Research Institute Travel Grant (2016)
David Walsh Student Prize (Best oral presentation in the fields of cell or developmental
biology by a student) – ComBio (2016)
Outstanding Student Oral Presentation Prize – 6th ANZSCDB Adelaide Meeting (2016)
Department of Molecular Cellular Biology Student Publication Prize - School of Biological
Sciences Research Day, The University of Adelaide (2016)
12
CHAPTER 1
Introduction
CHAPTER 1: Introduction
13
1.1 Epilepsy
Epilepsy is a neurological disease caused by a combination of genetic and environmental
factors and with 3-4% of the population developing the disorder at some stage in their life
this presents a serious burden to society. It is primarily defined by recurrent seizures and
while anti-epileptic drugs are an effective treatment for many patients, up to 30% of epilepsy
cases are drug refractory. Epileptic seizures can be the result of a genetic predisposition or a
structural abnormality in the brain e.g. a tumour or trauma. Seizure types can vary and are
important in classifying and managing an individual’s epilepsy. Partial seizures are limited
to only one region of the brain and can be termed “simple” if there is no loss of consciousness
and “complex” if consciousness is lost. Generalised seizures involve the whole brain and
consciousness is normally impaired. There are six major types of generalized seizures; tonic-
clonic, absence, myoclonic, tonic, clonic and atonic. The characteristics of each seizure type
vary depending on how they affect the neuronal networks in the brain. Understanding the
normal neuronal networks in the brain and how epilepsy alters their function is essential to
investigate the cause of epilepsy and for effective therapeutic intervention.
Identifying the causes of different epileptic disorders is essential to provide key information
that guides therapeutic treatment. Improvements in understanding the causes of epilepsy
have largely been due to advances in DNA sequencing technology. In 1975 Hauser and
Kurland estimated that the majority of epilepsy cases could be classified as “idiopathic”
(unknown cause) (Hauser and Kurland, 1975). Today, the term idiopathic has been left
behind and such cases are now thought to be monogenic syndromes (caused by mutation of
a single gene) or more genetically complex disorders which may involve modified
susceptibility loci (Figure 1-1) (Thomas and Berkovic, 2014).
Although single gene epilepsies are rare, investigating and understanding their molecular
causes provides important information regarding the molecular pathways and cellular
CHAPTER 1: Introduction
14
processes that are involved in causing or propagating seizures and in guidance for
pharmacotherapy and surgical intervention.
CHAPTER 1: Introduction
15
Figure 1-1. The majority of epilepsy cases are thought to be caused by a genetic predisposition.
Hauser and Kurland (1975) described most epilepsy cases as “idiopathic”. In recent years, with the
advances in DNA sequencing technology, it is thought that cases previously described as idiopathic
largely have a genetic basis. Adapted from Thomas and Berkovic (2014).
1975 (Hauser and Kurland) 2014 paradigm
CHAPTER 1: Introduction
16
1.2 Protocadherin 19 Girls Clustering Epilepsy
PROTOCADHERIN 19 Girls Clustering Epilepsy (PCDH19-GCE; also known as Epilepsy
and Mental Retardation limited to Females (EFMR)) is a female-specific condition caused
by mutation in the X-linked gene PCDH19 (Dibbens et al., 2008; Ryan et al., 1997). The
disorder is primarily defined by clusters of febrile seizures beginning in early childhood with
variable intellectual disability and autism spectrum disorder (ASD) (Marini et al., 2010;
Ryan et al., 1997; Scheffer et al., 2008). PCDH19-GCE is now recognised as the second
most common monogenic epilepsy disorder behind Dravet syndrome (Duszyc et al., 2014;
Marini et al., 2010). Symptoms in affected females vary from mild to severe, ranging from
benign focal epilepsy with normal cognitive function to severe seizures and intellectual
disability that resemble Dravet syndrome (Depienne and LeGuern, 2012; Scheffer et al.,
2008). Febrile seizures are the initial indicator in approximately half of all cases with seizure
types mostly involving generalized tonic, clonic or tonic-clonic and or focal seizures (Marini
et al., 2010).
PCDH19-GCE exhibits a unique X-linked mode of inheritance (Ryan et al., 1997). X-linked
diseases are usually characterized by affected males and unaffected carrier females (i.e. a
recessive mode of inheritance). In contrast, PCDH19-GCE only affects heterozygous
females, with transmitting hemizygous males showing normal cognitive function and no
seizures (Figure 2-1) (Depienne and LeGuern, 2012; Fabisiak and Erickson, 1990; Juberg
and Hellman, 1971; Ryan et al., 1997; Scheffer et al., 2008).
CHAPTER 1: Introduction
17
Figure 2-1. PCDH19-GCE displays a unique X-linked inheritance pattern. Most X-linked
disorders exhibit recessive inheritance in which hemizygous males are affected while heterozygous
female carriers are not (left). In contrast, PCDH19-GCE affects heterozygous females, while
hemizygous males are spared (right).
.
X-Linked Inheritance with Male Sparing
(PCDH19-GCE)
.
.
X-Linked Recessive
Inheritance
.
.
Unaffected
Carrier
Affected
CHAPTER 1: Introduction
18
Recent analysis of PCDH19-GCE individuals by multiple research groups has identified
over 100 mutations in the PCDH19 gene with both inherited and de novo mutations reported.
These include nonsense mutations, nucleotide insertions and deletions, mutations altering
the splice sites, missense mutations, intragenic and whole gene deletions (Figure 3-1)
(Depienne and LeGuern, 2012). The majority of pathological disease mutations are nonsense
and missense mutations positioned in exon 1, affecting highly conserved amino acids in the
extracellular domain of PCDH19. PCDH19 mRNAs with premature stop codons have been
shown to undergo nonsense mediated decay and together with identification of whole gene
deletions this suggests that loss of function is the likely outcome from the mutations which
has been confirmed through a series of bead aggregation assays (Cooper et al., 2016;
Dibbens et al., 2008). Furthermore, there does not appear to be any correlation between the
severity of the phenotype and the type of mutation i.e. the same mutation results in variable
degrees of disease severity in different individuals (Depienne et al., 2011; Higurashi et al.,
2013; Scheffer et al., 2008). This is further highlighted by different phenotypes presented in
a pair of monozygotic twins with PCDH19-GCE (Higurashi et al., 2013).
CHAPTER 1: Introduction
19
Figure 3-1. Schematic diagram highlighting the types and location of mutations leading to
PCDH19-GCE. Over 100 unique disease mutations have now been identified, the most common
being missense mutations positioned in exon 1. Adapted from Depienne and LeGuern (2012).
CHAPTER 1: Introduction
20
1.3 Pcdh19 expression in the mammalian brain
Pcdh19 was first discovered in 2001 when its mRNA expression was reported in multiple
tissues (Wolverton and Lalande, 2001). Pcdh19 expression is tightly controlled both
spatially and temporally within the central nervous system during mammalian development
(Figure 4-1) (Dibbens et al., 2008; Gaitan and Bouchard, 2006). In situ hybridisation of WT
Pcdh19 in mice indicates widespread expression in both the embryonic and postnatal CNS
including the cortex, hippocampus, retina, nasal epithelium and spinal cord (Dibbens et al.,
2008; Gaitan and Bouchard, 2006). Pcdh19 is highly expressed in the sub-ventricular zone,
intermediate zone, sub-plate, hippocampus, specific layers (II, IV, V and VI) of the cerebral
cortex and the subiculum. Expression in these specific regions suggests important functional
roles in neuronal development as well as potential roles in the formation of neuronal
networks and synaptogenesis.
CHAPTER 1: Introduction
21
Figure 4-1. Pcdh19 expression is widespread in the developing and postnatal mouse CNS. c)
High Pcdh19 mRNA levels were observed in multiple brain regions of sagittal 12.5 dpc embryo
sections. di, diencephalons; dmes, dorsal mesentery; ep, epiphysis; oe, olfactory epithelium; sc,
spinal cord. Scale bar 500 μm. Adapted from Gaitan and Bouchard (2006). g,h) Adjacent coronal
section through the mid-hippocampal region stained with haematoxylin and eosin and processed for
Pcdh19 in situ respectively. i) a more posterior brain section to h, showing Pcdh19 expression. j-l)
Higher-magnification images of the boxed regions in g and h. Arrowheads in j and k showing Pcdh19
expression within cortical layers II-IV. Cx-cortex. Scale bars in a,b,f-I 200μM, in c-e, j-I 50μM.
Adapted from Dibbens et al. (2008)
CHAPTER 1: Introduction
22
1.4 The Cadherin Superfamily
To comprehend how mutation of PCDH19 leads to human disease it is important to
understand its normal function. PCDH19 is a member of the protocadherin family, a
subgroup of the cadherin superfamily.
Cadherins are calcium-dependent cell-cell adhesion molecules that play major roles in
development, tissue morphogenesis, maintaining structural integrity of solid tissues and
regulating turnover and reorganization of tissue structures (Gumbiner, 1996, 2005; Hatta and
Takeichi, 1986; Hatta et al., 1988; Leckband and Prakasam, 2006; Patel et al., 2003;
Takeichi, 1995; Takeichi et al., 1981). They are single-pass transmembrane proteins defined
by multiple cadherin repeat sequences in their extracellular domain which mediate
homotypic cell-cell adhesion. The links between these repeats are made more rigid and
strengthened by the specific binding of Ca2+ ions (Boggon et al., 2002; Nagar et al., 1996).
The intracellular domains of the different cadherins are variable, allowing different
functional roles for each cadherin.
Cadherins are divided into three major subfamilies, the classical cadherins, desmosomal
cadherins and the protocadherins (Figure 5-1). Classical cadherins were the first group to be
discovered and can be split into two subfamilies; Type 1 and Type 2. They have common
features such as five extracellular cadherin repeats and conserved cytoplasmic domains that
interact with cytoskeletal proteins, most importantly α- and β-catenin. Absence of α- or β-
catenin leads to defective cell-cell adhesion and failure of cadherin-catenin complexes to
associate with the actin
CHAPTER 1: Introduction
23
Figure 5-1. Cadherins are divided into three main subgroups. Variation occurs in the number of
extracellular cadherin repeats present as well as the interaction domains in the cytoplasmic regions.
Adapted from Hirano and Takeichi (2012).
CHAPTER 1: Introduction
24
cytoskeleton (Hatta and Takeichi, 1986; Pettitt, 2005). Type 1 classical cadherins contain an
extracellular tryptophan residue (W2) and a corresponding hydrophobic pocket in the
cadherin repeat sequence, while type 2 classical cadherins contain two conserved tryptophan
residues (W2 and W4) and two hydrophobic pockets. The conserved tryptophan residues
and hydrophobic pockets are critical for both homodimerization and cell-cell adhesion
(Boggon et al., 2002). Desmosomal cadherins are highly similar to type 1 classical cadherins
but contain 4 extracellular repeats and have a different cytoplasmic domain allowing
connection to intermediate filaments. It is suggested that desmosomal cadherins are required
for strong cell-cell adhesion but is unclear whether this is primarily due to homophilic or
heterophilic interactions (Delva et al., 2009).
Protocadherins are structurally similar to classical cadherins however the conserved
tryptophan residues are replaced with two cysteine residues that are thought to perform a
similar function (Morishita et al., 2006). Currently more than 70 protocadherin genes have
been identified, making up the largest subgroup of the cadherin family. Protocadherins are
divided into two groups based on their genomic arrangement: clustered and non-clustered
(Figure 6-1). Clustered protocadherins are grouped within the genome, encoding 58
transcripts from only three gene clusters located on the same chromosome. Non-clustered
protocadherins are scattered throughout the genome and can be divided into two groups
PCDHδ and “other” protocadherins. Clustered protocadherins have six extracellular
cadherin repeats while members of the non-clustered family have between four and seven
cadherin repeats (Kim et al., 2011; Morishita and Yagi, 2007; Redies et al., 2005). There are
nine PCDHδ members, all of which contain two highly conserved regions (CM1 and CM2)
CHAPTER 1: Introduction
25
Figure 6-1. Clustered PCDHs consist of the α, β and γ subfamilies. Non-clustered PCDHs are
divided into two groups, PCDHδ family and other PCDHs. PCDHδ members are further
categorized by the presence/absence of a third highly conserved extracellular domain. Adapted from
Morishita and Yagi (2007).
Protocadherin Family
CHAPTER 1: Introduction
26
in their cytoplasmic domains (Morishita and Yagi, 2007). The PCDHδ family can be further
divided into two subgroups depending on; overall homology, number of EC repeats present
and conservation of specific amino acids within the cytoplasmic domain (Figure 6-1). The
PCDHδ1 subgroup consists of Pcdh-1, 7, 9 and 11, and the PCDHδ2 subgroup contains
PCDH-8, 10, 17, 18 and 19 (Morishita and Yagi, 2007). While the extracellular domain of
protocadherins is essential for homotypic cell-cell interactions (Morishita and Yagi, 2007;
Patel et al., 2003; Redies et al., 2005; Yagi and Takeichi, 2000) the cytoplasmic domains of
protocadherins are structurally diverse and may have the ability to form novel interactions.
Overall PCDHs have homotypic adhesion function which is regulated through the
extracellular domains, in contrast to the intracellular domains which are variable amongst
family members and are hypothesised to have unique functions.
CHAPTER 1: Introduction
27
1.5 Adhesive Functions of Protocadherins in the Mammalian Brain
Protocadherins have been reported to be implicated in many different processes within the
CNS including neuronal migration, organisation of different neuronal populations,
synaptogenesis, ectoderm differentiation, neuronal self-avoidance, cytoskeletal changes and
cell differentiation (Frank and Kemler, 2002; Hoshina et al., 2013a; Junghans et al., 2005;
Lefebvre et al., 2012; Morishita and Yagi, 2007; Redies et al., 2005).
In recent years the adhesive function of clustered PCDHs has been proposed to be crucial
for neuron identification of self from non-self within the mammalian brain, similar to the
function of DSCAM genes in Drosophila (Soba et al., 2007). Deletion of clustered Pcdhγ
genes disrupts the self-avoidance of neuronal processes in retinal starburst amacrine cells
and Purkinje cells (Figure 7-1A, left) (Lefebvre et al., 2012). Furthermore replacement of
the deleted Pcdhγ cluster with a single Pcdhγ isoform restores self-avoidance suggesting that
homophilic adhesion interactions between neurites from the same neuron generate a
repulsive signal leading to self-avoidance (Figure 7-1A, right) (Lefebvre et al., 2012). It has
been proposed that interactions between different neurons are allowed to occur as dendrites
will rarely encounter a matched set of clustered PCDHs, unless they originate from the same
soma (Lefebvre et al., 2012). Therefore, it is crucial that each neuron in the brain expresses
a unique combination of clustered PCDHs which is supported by the random expression
pattern of clustered PCDHs which is dependent on cis regulatory elements that determine
which members of the clusters are to be expressed (Ribich et al., 2006; Yokota et al., 2011).
For example, Purkinje cells express ~2 of the 5’ 12 Pcdhα isoforms, ~4 of the 5’ 19 Pcdhγ
members and ~4 of the 22 Pcdhβ isoforms while the 3’ members of the α and γ clusters are
expressed constitutively in all Purkinje cells (Yagi, 2012). This random pattern of expression
in
CHAPTER 1: Introduction
28
B
C
A
CHAPTER 1: Introduction
29
Figure 7-1. Clustered PCDHs have a functional role in neuronal self-avoidance which is
thought to arise by dictating specific interactions between cells. A) Deletion of all Pcdhγ
members in starburst amacrine cells leads to increased amount of “self” interactions. This can be
rescued by the expression of a single PCDHγ protein in PCDHγ null star amacrine cells. Adapted
from Lefebvre et al. (2012) B) K562 cells expressing the same clustered PCDH combinations display
extensive mixing. In contrast, mixing cells expressing different clustered PCDH combinations results
in segregation of green and red cells. Adapted from Thu et al. (2014). C) A model for PCDH mediated
cell-cell recognition based on lattice-like structures. A single mismatch of PCDH isoforms between
cells terminates chain extension. It is proposed that interaction of clustered PCDHs leads to an
avoidance event through an intracellular signalling. A repulsion signal is only achieved through the
formation of extensive PCDH lattice molecules. Mathematical modelling predicts that the size of the
PCDH lattice-like complex is dramatically reduced with just one PCDH mismatch between cells.
Adapted from Rubinstein et al. (2015).
CHAPTER 1: Introduction
30
Purkinje cells suggests that almost every neuron in the brain will contain a different profile
of clustered PCDH expression (Yagi, 2012).
The molecular logic of how clustered PCDHs determine self-avoidance has been
investigated through a series of cell aggregation and structural modelling experiments
(Goodman et al., 2016; Nicoludis et al., 2015, 2016; Rubinstein et al., 2015; Schreiner and
Weiner, 2010; Thu et al., 2014). Using K562 cells (which express no classical cadherins or
PCDHs) it was shown that homophilic interactions between different combinations of
clustered PCDHs are dependent on all isoforms expressed, such that a single mismatched
isoform disrupts adhesion of cells (Figure 7-1B) (Rubinstein et al., 2015; Schreiner and
Weiner, 2010; Thu et al., 2014). Structural studies by multiple groups revealed that EC1-
EC4 domains are critical for homophilic trans binding of PCDHs between adjacent cells and
that the EC6 domain is essential for heterophilic cis interactions of different PCDHs within
the same cell (Goodman et al., 2016; Nicoludis et al., 2015, 2016; Rubinstein et al., 2015).
Rubinstein et al (2015) proposed a model whereby clustered PCDHs form cis heterodimers
that engage in a head-to-tail homophilic trans interaction across two cells that form a zipper-
like structure. When two neurites from the same cell interact large zipper-like PCDH
complexes will form and lead to repulsive downstream signalling, but if two neurites from
different cells interact the zipper-like structure will terminate due to the mismatch in PCDHs
providing a size dependent mechanism for self-avoidance (Figure 7-1C) (Rubinstein et al.,
2015). It is thought that only large lattice structures will generate sufficient avoidance
signalling to cause neurite repulsion, in contrast to smaller mismatched lattice structures
where the avoidance signalling will be below the repulsion threshold.
The homophilic function of Pcdhγ isoforms has also been shown to regulate synapse
formation between distant neurons and deletion of the Pcdhγ cluster in cortical and
CHAPTER 1: Introduction
31
hippocampal neurons leads to simplified dendritic arbors (Garrett et al., 2012; Kostadinov
and Sanes, 2015; Suo et al., 2012). Furthermore, the homotypic adhesive function of Pcdhγ
molecules promotes dendritic arborisation at local sites between neurons and between
neurons and astrocytes (Molumby et al., 2016).
The degree of functional overlap between clustered and non-clustered PCDHs is unclear and
in particular whether PCDH19 and other non-clustered PCDHs act combinatorially has not
been addressed. Combinatorial activity of non-clustered PCDHs may be possible given that
the expression of non-clustered PCDHs spatially overlap in the developing and postnatal rat
CNS and individual neurons in the mouse somatosensory cortex have been shown to express
multiple non-clustered PCDH molecules (Figure 8-1) (Kim et al., 2007, 2010; Krishna-K et
al., 2011). Furthermore in vitro bead assays showed that PCDH19 interacts with N-cadherin
(N-CAD) to form a novel adhesion complex that cannot form trans interactions with
PCDH19 or N-CAD alone (Emond et al., 2011). This is reminiscent of the combinatorial
activity of the clustered PCDHs and supports the potential for PCDH19 to interact with
adhesion molecules including non-clustered PCDHs to form combinatorial adhesion
specificity.
Additionally, the non-stochastic nature of non-clustered PCDH expression suggests that
combinatorial adhesive codes dictated by this group of molecules will play roles in guiding
neurons to coalesce/form connections with other appropriate neurons, rather than providing
unique identity to each neuron.
CHAPTER 1: Introduction
32
Figure 8-1. Non-clustered PCDHs display overlapping expression in the developing and
postnatal rodent cortex. A) In situ hybridisation of rat embryos displays overlapping expression of
many non-clustered PCDHs in the CNS. Adapted from (Kim et al., 2007). B) Double fluorescent in
situ hybridisation of the mouse somatosensory cortex shows that multiple non-clustered PCDHs are
expressed within the same neuron. Adapted from Krishna-K et al. (2011).
A
B
CHAPTER 1: Introduction
33
1.6 Neural Function of PCDH19
To date the endogenous function of PCDH19 has not been investigated in a mammalian
brain, primarily being limited to studies performed in vitro and using the model organism
Danio rerio (zebrafish).
Cell culture assays have been utilised to show PCDH19 can cause calcium dependent cell
aggregation (Tai et al., 2010). Additionally, cells expressing PCDH19 were labelled and
mixed with cells expressing PCDH10 and both populations formed aggregates
predominantly containing either PCDH19 or PCDH10 cells, confirming the homotypic
adhesion function of PCDH19 (Tai et al., 2010). The function of endogenous PCDH19 has
been investigated in zebrafish using knock down and knock out approaches (Cooper et al.,
2015; Emond et al., 2009). Unfortunately, these studies provided conflicting results making
it difficult to interpret the role of PCDH19 in the CNS. Morpholino injections targeting
pcdh19 resulted in a significant knockdown of pcdh19 gene expression (Emond et al., 2009).
Morphant embryos exhibited a misfolded midbrain-hindbrain region compared to wild type
embryos at 28 hours post fertilisation. At the 10 somite stage pcdh19 morphants exhibited
malformations of the anterior neural rod, displayed impaired convergent cell movements in
the lateral plate and a significantly wider neural plate. Knockdown of pcdh19 also resulted
in cell motility defects in the anterior neural plate including a decrease in the directional
specificity and a reduced level of coupled migration with neighbouring cells. A second study
deleted pcdh19 in zebrafish using TALEN genome editing technology (Cooper et al., 2015).
In contrast to the above mentioned study, Pcdh19 null zebrafish were viable and fertile and
did not display any defects in neural plate morphology. However, disruption of columnar
organisation in the optic tectum was observed leading to defects of visually guided
behaviours. These data suggest a role for Pcdh19 in organisation of the developing nervous
system. However, the conflicting phenotypes resulting from knock down versus knock out
CHAPTER 1: Introduction
34
experiments makes it difficult to interpret the endogenous role of Pcdh19 in the mammalian
brain and to gain insight into how mutation leads to disease.
The adhesive roles of multiple non-clustered PCDHs in the mammalian brain have been
investigated using genetically modified mice (Hayashi et al., 2014; Hoshina et al., 2013b;
Uemura et al., 2007; Yamagata et al., 1999; Yasuda et al., 2007). Pcdh17 null mice have
been shown to have defects in both the regulation of presynaptic assembly in corticobasal
ganglia circuits and abnormalities in axon projections from the amygdala to the
hypothalamus (Hayashi et al., 2014; Hoshina et al., 2013a). Similarly, Pcdh10 deficient
mice have defects in axonal pathways through the ventral telencephalon (Uemura et al.,
2007). Studies of Pcdh7 and Pcdh8 in mice have revealed their roles in retinal ganglion cell
dendrite arborisation and disruption of axonogenesis and the regulation of dendritic spine
density and synaptogenesis, respectively (Leung et al., 2013; Piper et al., 2008). The
cytoplasmic domains of PCDH17, PCDH10 and PCDH8 are also likely to play a role in axon
guidance as they have been shown to interact with the WAVE regulatory complex which
controls actin cytoskeletal dynamics (Chen et al., 2014). PCDH19 has also been reported to
interact with WAVE complex proteins and therefore could also be implicated in the process
of axon guidance (Chen et al., 2014).
As non-clustered PCDHs are highly conserved amongst members these studies provide us
with insights into possible functions of PCDH19 in the mammalian brain.
CHAPTER 1: Introduction
35
1.7 What causes the unusual inheritance of PCDH19-GCE?
The most striking characteristic of PCDH19-GCE is its unique pattern of X-linked
inheritance. This feature is poorly understood as current model organism studies have been
limited to zebrafish where pcdh19 is positioned on chromosome 14. To date multiple
theories have been proposed to explain the unique pattern of inheritance but there is a lack
of supporting evidence.
Given that males with PCDH19 mutations (hemizygous) do not develop seizures and have
normal cognitive function, it is suggested that the complete loss of PCDH19 is not
pathogenic. On the other hand, females with one wild type and one mutated allele of
PCDH19 (heterozygous) are affected. Therefore it was initially proposed that PCDH19
mutations were dominant negative, but the identification of whole gene deletions and cases
of nonsense mediated decay provided strong evidence opposing this hypothesis (Dibbens et
al., 2008). It was also suggested that males may be able to compensate for the loss of
PCDH19 through the Y-linked PCDH11Y. However, this has also been ruled out due to the
discovery of several mosaic males with early somatic mutations in PCDH19, that phenocopy
affected girls with heterozygous mutations (Depienne et al., 2009; Terracciano et al., 2016).
The current withstanding hypothesis proposes that the coexistence of WT and mutant
PCDH19 neurons arising through random X-inactivation underlie the unique pattern of
inheritance (Figure 9-1) (Depienne et al., 2009; Dibbens et al., 2008). It is suggested that
this mosaicism leads to abnormal neuronal connections between PCDH19 WT and PCDH19
mutant cells affecting neural network formation. However, it remains to be determined how
mosaicism could lead to PCDH19-GCE at the molecular, cellular and network level.
Furthermore, this is unable to explain how the complete absence of PCDH19 does not result
in epilepsy.
CHAPTER 1: Introduction
36
Figure 9-1. PCDH19-GCE is proposed to be caused by abnormal connections between PCDH19
positive and PCDH19 negative neurons. A) Normal individuals will have one population of
PCDH19 containing cells and will be able to form proper neural networks. B) Similarly, hemizygous
males will have population of PCDH19 negative cells and will form normal neuronal connections
like normal individuals. C) Heterozygous females have two populations of cells, PCDH19 positive
and PCDH19 negative, which cannot interact with each other to form appropriate neural networks,
leading to disease. Adapted from Depienne et al. (2009).
Normal Individual (male or female)
PCDH19 positive cells only
Mutated Males
PCDH19 negative
cells only
Mutated Females and Mosaic Mutated Males
PCDH19 positive cells and
PCDH19 negative cells co-exist
Asymptomatic Asymptomatic PCDH19-GCE
CHAPTER 1: Introduction
37
1.8 Pcdh19 Null Mouse
Genetically manipulated mice are commonly used to understand how genetic mutations
contribute to disease phenotypes. By knocking in or knocking out genes implicated in
disease the functional role proteins play in disease and the phenotype can be studied, leading
to better understanding of the disease and potential new treatments for humans.
As Pcdh19 is also X-linked in mice a suitable approach to investigate to the cellular and
molecular basis of PCDH19-GCE would be to generate a mouse model in which Pcdh19 is
mutated to mimic a human PCDH19-GCE mutation. However, this approach assumes that
the pathogenic mechanism underpinning PCDH19-GCE is conserved in mouse and human.
Epilepsy has an obvious seizure phenotype providing an accessible target to study as a
complex trait in experimental animal models. Several examples of laboratory mice exist that
model epilepsy, including Cntnap2 deficient mice, which model temporal lobe epilepsy
(Buckmaster and Lew, 2011; Peñagarikano et al., 2011). Spontaneous mouse models of
epilepsy also exist in the form of DBA mice which experience severe tonic-clonic seizures
in response to audiogenic stimuli (Hall, 1947) and the EL mouse strain which has limbic and
tonic-clonic convulsions that are induced by routine animal handling (Rise et al., 1991;
Suzuki and Yurie, 1977).
With this in mind we acquired a potential disease mouse model for PCDH19-GCE. Lexicon
Pharmaceuticals engineered a mouse where exon 1,2 and 3 of the Pcdh19 gene are replaced
with a β-galactosidase/neomycin fusion cassette as selection markers. Exon 1, 2 and 3
encode the entire extracellular and transmembrane region so by deleting these regions it
should result in loss of PCDH19 function. Since it is thought that PCDH19-GCE mutations
are loss-of-function it was hypothesised that the Pcdh19 mutant mouse could be used to
model PCDH19-GCE and gain insight into the cellular and molecular cause of the disease.
CHAPTER 1: Introduction
38
1.9 Project Rationale
PCDH19 is a homotypic cell-cell adhesion molecule, which when mutated causes epilepsy
and cognitive impairment in humans. PCDH19-GCE has a unique X-linked inheritance
pattern whereby heterozygous females are affected and hemizygous males are not. It has
been proposed that abnormal neuronal interactions occur between PCDH19 WT and
PCDH19 mutant cells in the brain of heterozygous females leading to disease. However, the
molecular and cellular mechanism causing disease is unknown. Furthermore, it is not
understood how the complete absence of PCDH19 in hemizygous males does not lead to
pathology. These are the key questions that will be investigated in the research of this thesis,
with the aims of:
1. Characterising the endogenous function of PCDH19 in the mammalian brain.
2. Investigating the cellular mechanism that underlies the unique inheritance pattern of
PCDH19-GCE.
39
CHAPTER 2
Pcdh19 Loss-of-Function Increases
Neuronal Migration In Vitro but is
Dispensable for Brain Development
in Mice
Daniel T. Pederick, Claire C. Homan, Emily J. Jaenhe, Sandra G. Piltz, Bryan
P. Haines, Bernhard T. Baune, Lachlan A. Jolly, James N. Hughes, Jozef
Gecz and Paul Q. Thomas
Published in: Scientific Reports 2016. 6, 26765.
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable for
Brain Development in Mice
40
2.1 Summary
Using cultured cell assays PCDH19 has been shown to have weak homotypic cell-cell adhesion
activity, interact with N-cadherin to form novel adhesion complexes and interact with various
members of the WAVE complex (Emond et al., 2011; Tai et al., 2010). To understand the
endogenous role of PCDH19 in the CNS the majority of studies have used zebrafish as a model
organism. Two independent studies using morpholino knockdown of pcdh19 or TALEN
mediated null zebrafish gave severe and mild phenotypes, respectively (Cooper et al., 2015;
Emond et al., 2009). These conflicting data make it difficult to interpret the endogenous
function of PCDH19 in the CNS. Furthermore, the endogenous role of PCDH19 in the
mammalian brain has yet to be investigated and therefore was addressed in the paper presented
in this chapter entitled “Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is
Dispensable for Brain Development in Mice”.
In this paper we validated the first Pcdh19 null allele in mice confirming the expression of
PCDH19 protein in the hippocampus, cortex and cerebellum. We also showed that the β-
Galactosidase/Neomycin reporter allele, marking Pcdh19 null cells, was expressed in the same
brain regions. We confirmed the expression of PCDH19 in the synapses of hippocampal
neurons and ruled out the presence of gross morphological defects in the brains of Pcdh19
heterozygous and homozygous null mice. We also showed that Pcdh19 null cells were not
ectopically located in the developing and postnatal brains of Pcdh19 heterozygous and
homozygous null mice. Furthermore, we showed a subtle increase in neuronal migration
potential of Pcdh19 null cells in vitro although this did not translate to overt positional changes
of neurons in the developing and adult mouse brain. While further detailed analysis of this
mouse model may reveal subtle defects in Pcdh19 null mouse brains, overall we show that
despite widespread expression of Pcdh19 in the CNS, deletion of Pcdh19 does not grossly
affect brain develop
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable for
Brain Development in Mice
41
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable for
Brain Development in Mice
42
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable for
Brain Development in Mice
43
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
44
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
45
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
46
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
47
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
48
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
49
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
50
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
51
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
52
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
53
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
54
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
55
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
56
CHAPTER 2: Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable
for Brain Development in Mice
57
58
CHAPTER 3
Abnormal cell sorting is associated
with the unique X-linked
inheritance of PCDH19 epilepsy
Daniel T. Pederick, Kay L. Richards, Sandra G. Piltz, Simone A. Mandelstam, Russell C.
Dale, Ingrid E. Scheffer, Jozef Gecz, Steven Petrou, James N. Hughes and Paul Q.
Thomas
Submitted to: Science on 27.1.2017
CHAPTER 3: Abnormal cell sorting is associated with the unique X-linked inheritance of PCDH19
epilepsy
59
3.1 Summary
The most intriguing characteristic of PCDH19-GCE is its unique pattern of X-linked
inheritance. To date there is no experimental evidence providing insight into the molecular
and cellular mechanisms underlying the unique inheritance. Therefore, this was addressed
in the submitted manuscript presented in this chapter entitled “Abnormal cell sorting is
associated with the unique X-linked inheritance of Protocadherin 19 epilepsy”.
To begin investigating the cellular mechanism that underlies the unique X-linked inheritance
of PCDH19-GCE we used an in vitro system to investigate the role of PCDH19 in dictating
specific cell-cell interactions and how disease causing missense mutations impact normal
function. K562 cells were used to perform these experiments as they do not express
endogenous protocadherins or classical cadherins (Ozawa and Kemler, 1998; Schreiner and
Weiner, 2010), meaning all cell-cell interactions we observed were dependent on the PCDHs
we exogenously expressed.
Two populations of cells co-expressing the same combination of NC PCDHs displayed
extensive mixing but when one of these populations had a NC PCDH deleted, added or
substituted, the two cell populations displayed significant sorting into two groups dependent
on the NC PCDHs they expressed. We also performed aggregation assays using K562 cells
and confirmed that three disease causing missense mutations lack adhesive activity. We next
mixed cells expressing WT PCDH19/PCDH10 with mutant PCDH19/PCDH10 and
observed significant segregation of the two cell populations. These experiments provide the
first evidence that NC PCDHs can cooperate to determine adhesion specificities which are
sensitive to single PCDH differences.
Next, we sought to investigate how perturbation of these adhesion codes would manifest in
the developing mammalian brain where complex arrays of adhesion molecules direct
CHAPTER 3: Abnormal cell sorting is associated with the unique X-linked inheritance of PCDH19
epilepsy
60
morphogenesis. We showed that young adult heterozygous mice display abnormally
elevated neuronal activity and that this phenotype is not present in homozygous mice,
consistent with the unique X-linked inheritance of PCDH19-GCE in humans.
To determine the impact of mosaic PCDH19 expression at the cellular level in vivo we
required reporter alleles for Pcdh19-expressing WT and null cells. To label Pcdh19 null
cells, we used the previously validated Pcdh19 null mice from Chapter 2. In Chapter 3 we
describe the generation of a mouse model allowing unequivocal identification of PCDH19
WT cells. Specifically, we utilised CRISPR-Cas9 genome engineering to insert a HA-FLAG
epitope tag at the C-terminus of PCDH19 allowing detection of endogenous protein using
commercially-available antibodies. We show for the first time PCDH19 spatial expression
which was widely expressed in the developing brain, becoming restricted in the postnatal
cortex.
To investigate the phenotype resulting from disruption of PCDH19-dependent cell adhesion
codes, we generated female mice with both Pcdh19HA-FLAG and Pcdh19 null alleles.
Simultaneous staining with HA antibodies and X-gal revealed striking segregation of
PCDH19-positive and PCDH19-null cortical neuroprogenitors. Abnormal cell sorting
observed in heterozygous mice demonstrates that mosaic expression of PCDH19 generates
distinct cell populations with incompatible adhesion codes. Furthermore, we show that the
cell sorting phenotype could be removed by deleting the remaining PCDH19 WT allele in
heterozygous female embryos. This confirms that the complete absence of PCDH19 rescues
abnormal cell sorting and provides a clear cellular phenotype that is associated with
PCDH19-GCE.
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epilepsy
61
Finally, analysis of MRI scans from a cohort of PCDH19-GCE patients revealed variable
cortical folding abnormalities in four patients with commonly occurring PCDH19-GCE
mutations.
Overall Chapter 3 describes how the mosaic expression of Pcdh19 leads to the generation of
differential adhesion codes in neural progenitor cells and appears to be the underlying
cellular mechanism responsible for the unique X-linked inheritance pattern of PCDH19-
GCE.
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epilepsy
62
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epilepsy
63
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epilepsy
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Submitted Manuscript: Confidential
Title: Abnormal cell sorting is associated with the unique X-linked inheritance
of PCDH19 Epilepsy
Authors: Daniel T. Pederick1, 2, Kay L. Richards3, Sandra G. Piltz1, 2, Simone A. Mandelstam4, 5, 6,
Russell C. Dale7, Ingrid E. Scheffer3, 8, Jozef Gecz1, 2, 9, 10, Steven Petrou3, James N. Hughes1, 2 and
Paul Q. Thomas1, 2, 10*
Affiliations:
1School of Biological Sciences, The University of Adelaide, Adelaide, South Australia 5005,
Australia
2Robinson Research Institute, The University of Adelaide, Adelaide, South Australia 5005, Australia
3Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Melbourne,
Victoria 3010, Australia
4Department of Paediatrics, The University of Melbourne, Melbourne, Victoria 3010, Australia
5Department of Radiology, The University of Melbourne, Melbourne, Victoria 3010, Australia
6Department of Medical Imaging, Royal Children's Hospital, Florey Neurosciences Institute,
Parkville, Victoria 3052, Australia
7Institute for Neuroscience and Muscle Research, University of Sydney8University of Melbourne,
Austin Health and Royal Children’s Hospital, Victoria, 3084, Australia
9School of Medicine, The University of Adelaide, Adelaide, South Australia 5005, Australia
10 South Australian Health and Medical Research Institute, Adelaide, South Australia 5000, Australia
*Correspondence to: [email protected]
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epilepsy
67
Abstract:
X-linked diseases typically exhibit more severe phenotypes in males than females. In contrast,
Protocadherin 19 (PCDH19) mutations cause epilepsy in heterozygous females but spare
hemizygous males. The cellular mechanism responsible for this unique pattern of X-linked
inheritance is unknown. We show that PCDH19 contributes to highly specific combinatorial
adhesion codes such that mosaic expression of Pcdh19 in heterozygous female mice leads to striking
sorting between WT PCDH19- and null PCDH19-expressing cells in the developing cortex,
correlating with altered network activity. In addition, we identify variable cortical malformations in
PCDH19 epilepsy patients. Our results highlight the role of PCDH19 in determining specific
adhesion codes during cortical development and how disruption of these codes is associated with the
unique X-linked inheritance of PCDH19 epilepsy.
One Sentence Summary: A single difference in PCDH expression disrupts complex adhesion codes
present within the developing mammalian cortex.
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epilepsy
68
Main Text:
Protocadherins (PCDHs) are the largest family of adhesion molecules and regulate axon
guidance/sorting, neurite self-avoidance and synaptogenesis (1–3). Mutations in PCDH
family members have been associated with a variety of neurological disorders including
epilepsy, autism and schizophrenia (4–7). Notably, mutations in Protocadherin 19
(PCDH19) cause PCDH19 Girls Clustering Epilepsy (PCDH19-GCE) which is reported to
be the second most common cause of monogenic epilepsy (8, 9). PCDH19-GCE is an X-
linked disorder with a unique pattern of inheritance whereby heterozygous females are
affected while hemizygous males are spared (8, 10). It has been proposed that the coexistence
of WT and mutant PCDH19 neurons that arise through random X-inactivation underpins the
unique inheritance (8, 11). This hypothesis is supported by the existence of affected males
who are mosaic carriers of somatic PCDH19 mutations (11). However, it is currently unclear
what processes are regulated by PCDH19 in the brain and how these are disrupted by mosaic
expression but not by the complete absence of functional protein.
PCDH19 has been shown to function as a homotypic cell adhesion molecule in vitro (12)
and recently published data indicate that PCDH19 has a virtually identical structure to
members of the closely-related clustered PCDH family (13). Interestingly, clustered PCDHs
have been shown to form combinatorial adhesion codes that are likely to play a role in
vertebrate neuronal self-avoidance (14–16). Given the overlapping expression of non-
clustered (NC) PCDHs in vivo (17, 18), we hypothesized that PCDH19, in combination with
other NC PCDH family members, could contribute to complex adhesion codes and that
perturbation of these codes underpins the unique X-linked inheritance pattern of PCDH19-
GCE.
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epilepsy
69
To investigate the adhesion activity of PCDH19 in combination with other NC PCDHs we
performed mixing experiments using K562 cells which ordinarily do not aggregate during
culture due to a lack of endogenous PCDHs and classical cadherins (16, 19). PCDH17 and
PCDH10 were selected for these experiments due to their ability to interact in cis with
PCDH19 when co-expressed in the same cell in a co-immunoprecipitation assay (Fig. 1A)
and overlapping expression with Pcdh19 in the embryonic mammalian brain (17). K562
cells transfected with different fluorescently-labelled PCDH combinations were mixed in a
1:1 ratio and quantitatively assessed for the presence/absence of sorting between the two
populations (Fig. 1B). When both cell populations expressed the same combination of
PCDHs random mixing was observed (Fig. 1C). In contrast, populations expressing different
PCDH combinations exhibited significant cell sorting indicating that cells with matching
PCDH profiles preferentially adhere (Fig. 1C). Notably, significant sorting occurred
between populations that differed by a single PCDH. We next assessed the impact of
PCDH19-GCE missense mutations on combinatorial PCDH adhesion. We first individually
expressed PCDH19 containing one of three commonly occurring missense mutations in
K562 cells and found that each lacked adhesive function (Fig. S1), consistent with previously
reported data for other missense mutations (13). When PCDH10 was co-expressed with
either WT PCDH19 or mutant PCDH19.N340S, significant sorting occurred between the
two populations (Fig. 1C). Taken together, these data suggest that PCDH19 forms
heterotypic cis interactions with NC PCDHs and contributes to combinatorial adhesion codes
that are sensitive to single PCDH differences.
Next, we sought to investigate how perturbation of these adhesion codes would manifest in
the developing mammalian brain where complex arrays of adhesion molecules direct
morphogenesis. For these experiments we used mice carrying a null allele (Pcdh19β-Geo)
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70
(20) to generate female heterozygous mice with mosaic expression of Pcdh19. Given the
seizures and elevated neural activity of PCDH19-GCE affected females (21, 22), we initially
performed electrocorticogram (ECoG) analysis on young adult mice to investigate if a
phenotype exists in heterozygous mice that does not manifest in homozygote animals. ECoG
traces from Pcdh19+/β-Geo (+/β-Geo) postnatal day 42 (P42) mice showed a consistent
increase in amplitude compared to Pcdh19+/+ (+/+) and Pcdh19β-Geo/β-Geo (β-Geo/β-Geo)
animals, which were themselves indistinguishable (Fig. 2A). Strikingly, ECoG signatures
for +/β-Geo mice showed a significant increase in mean number of spike-wave discharge
(SWD) events per hour and event duration compared with +/+ mice (Fig. 2B, C and S2). In
contrast, the mean number of SWDs per hour and the mean duration of a SWD event for β-
Geo/β-Geo animals was not significantly different to +/+ mice (Fig. 2B, C and S2). This
indicates that mosaic expression of Pcdh19 in mice results in altered brain network activity.
In contrast, this phenotype is not present in mice completely lacking PCDH19, consistent
with the unique X-linked inheritance of PCDH19-GCE in humans.
To determine the impact of mosaic PCDH19 expression at the cellular level in vivo we
required reporter alleles for Pcdh19-expressing WT and null cells. To label Pcdh19-
expressing null cells, we used the Pcdh19β-Geo β-galactosidase knock-in reporter allele,
which we had previously validated using X-Gal staining (20). To enable unequivocal
identification of WT PCDH19-expressing cells, we employed CRISPR-Cas9 genome editing
to insert an HA-FLAG epitope sequence at the C-terminus of the PCDH19 ORF (Fig. S3A).
Successful insertion was validated via PCR, sequencing, western blot and HA
immunohistochemistry (Fig. S3B, C and D, respectively). Consistent with previously
published mRNA in situ hybridization data (8, 20, 23), we detected PCDH19 expression in
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71
many brain regions, including prominent expression in the developing cortex that became
more restricted in the postnatal brain (Fig. S3D, E and S4).
Simultaneous labelling of PCDH19 positive (HA) and negative (X-gal) cells in Pcdh19HA-
FLAG/β-Geo (HA-FLAG/β-Geo) brains enabled us to investigate the phenotype resulting from
disruption of PCDH19-dependent cell adhesion codes. HA immunostaining of HA-FLAG/β-
Geo brains revealed a striking alternating pattern of PCDH19-positive and PCDH19-
negative cells that extended from the ventricular zone to the cortical plate. (Fig. 2D, left). X-
gal staining of HA-FLAG/β-Geo brains revealed a complementary and non-overlapping
pattern to HA immunostaining (Fig. 2D, left), suggesting this pattern is due to segregation
of PCDH19-positive and PCDH19-null cells To confirm that this pattern did not arise due
to random X-inactivation and subsequent clonal expansion, we examined HA-staining in
“wild type” Pcdh19HA-FLAG/+ (HA-FLAG/+) control embryos (Fig. 2D, right). This allowed
us to identify the pattern of PCDH19-positive HA labelled cells in the presence of PCDH19-
positive unlabelled cells. Within the cortex, X-inactivation manifested as small interspersed
patches of HA-positive and HA-negative staining along the ventricle and overlying neural
progenitors in the ventricular zone, with only subtle variations in HA staining within the
cortical plate (Fig. 2D, right). The significantly increased percentage of HA-negative regions
in HA-FLAG/β-Geo embryos compared with HA-FLAG/+ controls confirms that PCDH19-
positive and PCDH19-null cells coalesced into distinct groups (Fig. 2E). We also noted
significantly increased variation of HA-immunostaining across different HA-FLAG/β-Geo
animals, suggesting differences in abnormal cell sorting phenotypes are due to unique
patterns of X-inactivation in each embryo (Fig. 2F, S5 and S6). Consistent with an active
role in cell sorting, PCDH19 was present at interfaces between PCDH19 expressing radial
glial cells but not between PCDH19-positive and negative cells, supporting its role as a
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homotypic cell-cell adhesion molecule in vivo (Fig. S7). Cell body redistribution was
independently confirmed by staining for nuclear-localised SOX3 in Pcdh19+/β-Geo; Sox3+/-
trans-heterozygous mice (Fig. S8). Taken together, these data suggest that mosaic expression
of Pcdh19 in neuroprogenitors generates distinct cell populations with incompatible
adhesion codes that abnormally segregate during cortical development.
The absence of pathology in hemizygous males and the normal brain activity of mice
completely lacking functional PCDH19 (Fig. 2A, B and C) suggests that the removal of
differential adhesion codes dictated by mosaic PCDH19 expression will restore normal cell
sorting during cortical development. To test this hypothesis we developed a method to assess
in vivo cell sorting by visualising Pcdh19 allele-specific expression in functionally null
PCDH19 female mice. Using CRISPR-Cas9 genome editing, we deleted the Pcdh19HA-FLAG
allele in HA-FLAG/β-Geo zygotes to create DEL/β-Geo null embryos (Fig. 2F). Deletion of
exon 1 and lack of functional PCDH19 was validated by sequencing and HA
immunostaining (Fig. 2G and data not shown). X-gal staining of DEL/β-Geo embryos at 14
days post embryo transfer demonstrated that the two populations of null cells readily
intermix, thereby rescuing the abnormal cell sorting phenotype observed in HA-FLAG/β-
Geo embryos (Fig. 2G). Quantification of X-gal staining variation confirmed that the
abnormal cell sorting was rescued in DEL/β-Geo cortices (Fig. 2I and S4). This provides
further evidence that the differential adhesion codes of PCDH19-positive and PCDH19-
negative cells leads to abnormal cell sorting. Importantly, the lack of abnormal cell sorting
in DEL/β-Geo null embryos provides a clear cellular phenotype that correlates with and
likely explains the unique X-linked inheritance of PCDH19-GCE.
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Given that cortical neuroprogenitor cells predominantly undergo radial as opposed to lateral
movement (24), the presence of large blocks of cells in HA-FLAG/β-Geo embryos at 14.5dpc
suggests that abnormal cell sorting must occur at an earlier stage in development. We were
also interested to determine whether abnormal cell sorting persists in post-mitotic neurons.
Initially, we established that PCDH19 expression in the developing brain commences at 9.5
dpc (Fig. S9). Comparison of HA-FLAG/+ and HA-FLAG/β-Geo embryos at 9.5 dpc
revealed a similar pattern of highly interspersed PCDH19-positive and -negative cells (Fig.
3A) indicating that segregation had not yet occurred. In contrast, at 10.5 dpc abnormal cell
sorting was evident in HA-FLAG/β-Geo embryos but not in HA-FLAG/+ controls (Fig. 3A).
In addition, abnormal cell sorting was observed in postnatal neurons that express PCDH19
(Fig. 3B). The rapid onset and persistence of abnormal cell sorting demonstrates that cortical
progenitors are acutely sensitive to disruptions in specific adhesion codes and that these
embryonic cellular rearrangements are maintained in postmitotic neurons.
Although central nervous system (CNS) abnormalities have not previously been described
in +/β-Geo mice (20) we hypothesized that abnormal cell sorting caused by mosaic
expression of PCDH19 during human cortical development could result in morphological
defects due to the prolonged expansion of neuroprogenitors and extensive sulcation relative
to mice (25). Using data from the Allen Brain Atlas we confirmed PCDH19 is highly
expressed in human embryonic CNS including the cortex during the key neurogenic period
of 8-16 weeks post conception (26, 27) (Fig. S10A). PCDH19 expression is reduced
postnatally, but is still detectable, with the brain remaining the predominant site of
expression in the adult (Fig. S10B). We then reviewed MRI images from a cohort of
PCDH19-GCE girls. We identified abnormal cortical sulcation in four patients with common
causative mutations in PCDH19 (p.N340S, p.S671X and p.Y366LfsX10; Fig.4, S11 and
Table S2) (21). The patients presented with variably positioned cortical defects that included
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bottom of the sulcus dysplasias, abnormal cortical folding, cortical thickening and blurring
of the grey/white junction. These data suggest that subtle CNS abnormalities are a feature of
PCDH19-GCE, which is supported by previous observations of dysplastic neurons (10).
While it is not known how abnormal cell sorting could generate these defects, their
variability is consistent with the random nature of X-inactivation and phenotypes observed
in PCDH19-GCE patients.
In summary, the generation of differential adhesion codes in neural progenitor cells caused
by mosaic expression of Pcdh19 appears to be the underlying cellular mechanism
responsible for the unique X-linked inheritance pattern of PCDH19-GCE. Furthermore, our
data suggests that uniform removal of PCDH19, as seen in hemizygous males, does not lead
to the formation of incompatible adhesion codes allowing for normal positioning of
neuroprogenitor cells and neural activity (Fig. S12). The abnormal rearrangement of
neuroprogenitor cells in the incipient cortex of heterozygous brains indicates that at least
some of their neuronal progeny will be aberrantly positioned, regardless of whether they
maintain expression of Pcdh19 postnatally. This rearrangement has the potential to perturb
functional boundaries within the cortex and alter connectivity between cortical and
subcortical regions.
More broadly, our data suggests that neurodevelopmental disorders associated with mutation
of other NC PCDH family members (4–7) may be caused by disruption of adhesion codes.
Since all other NC PCDHs are autosomal, mosaic disruption of adhesion codes would have
to occur through a process other than X-inactivation. There is strong evidence that NC
PCDHs are subject to random monoallelic expression (RMAE) (28). Individuals with a
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heterozygous germ line mutation in a given autosomal NC PCDH would have a proportion
of their cells expressing either the functional or non-functional allele, resulting in disrupted
adhesion codes. The proportion of RMAE affected cells would likely impact the penetrance
or expressivity of any resultant phenotype.
Although both clustered and NC PCDHs can function in combinatorial adhesion complexes,
our data indicates that the interaction of matching adhesion codes for each of these closely-
related protein families in vivo can lead to different outcomes. Matching codes of clustered
PCDHs are thought to result in repulsion which has been implicated in neuronal self
avoidance. In contrast, our data indicate that cells expressing matching NC PCDH codes
selectively associate in vivo. Given the complex and overlapping expression pattern of NC
PCDHs throughout development it seems likely that this property may direct spatial
positioning of neuroprogenitors and could conceivably be utilized during morphogenesis of
other organs.
The ability of cells with different identities to self-associate and form discrete populations
was first identified in the early 1900s by mixing sponge cells from different colored species
(29–31). We now appreciate the critical role of adhesion molecules in regulating cell identity
and directing tissue morphogenesis in many developmental contexts including in the brain
(32–35). Our findings advance this field by providing evidence that perturbation of cellular
adhesion codes underlies the unique X-linked inheritance pattern of PCDH19-GCE and
suggests that just a single difference in PCDH expression is enough to disrupt the complex
adhesion codes present within the developing mammalian cortex.
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Acknowledgments: We thank Dr L. Luo (Stanford University) and members of the Thomas
lab for discussion and comments on the manuscript. We thank the patients and their families
for participating in our research, together with their referring clinicians. D.T.P was supported
by an Australian Government Research Training Program Scholarship. This work was
supported by an NHMRC Program Grant (400121) and the PCDH19 Alliance. The
supplementary materials contain additional data. Author contributions: D. T. P., J.G., J. N.
H. and P. Q. T. conceived and designed the study; D.T.P. performed and analysed all
experiments except the ECoG recordings which were performed by K. L. R. and S. P; S. G.
P. performed the microinjection of mouse zygotes; recruitment and phenotyping of the
patient cohort was performed by R.C.D. and I. E. S. and S. A. M. analysed the MRI images.;
D. T. P. prepared all figures; D. T. P. , J. N. H., and P. Q. T. wrote the manuscript; and all
authors revised the manuscript.
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Fig. 1. δ2 non-clustered PCDHs form combinatorial codes which dictate adhesion specificity.
(A) Co-immunoprecipitation of K562 cell lysates expressing PCDH combinations indicated by the
+ symbol. Lysates were pulled-down with FLAG Ab and subsequent blotting was performed with
MYC Ab (N=3 experiments). Cis interaction of FLAG-PCDH19 with MYC-PCDH19, MYC-
PCDH17 and MYC-PCDH10 was detected. Cells transfected with PCDHγC3-FLAG and Ncad-
MYC served as a negative control for cis-interaction as previously reported (14). (B) Schematic
diagram describing mixing assays performed to assess adhesion specificity. Fluorescently-labelled
K562 cells expressing different NC PCDH combinations were mixed in a 1:1 ratio, allowed to
aggregate and quantitatively assessed for the degree of mixing/sorting between the two populations.
(C) Quantification of the degree of mixing using Pearson’s correlation coefficient. Expression
constructs are indicated by 19 (PCDH19), 17 (PCDH17), 10 (PCDH10) and 19.N340S (PCDH19
N340S missense mutation) (***P < 0.001, ****P < 0.0001, ns=not significant, one-way analysis
of variance (ANOVA) with Tukey’s multiple comparison test against 19/10 +19/10 unless otherwise
indicated). Representative images of quantified K562 mixing assays. Cell sorting is observed when
populations differ by just one NC PCDH (panels (2), (3), (4), (5)), in contrast to mixing which occurs
in populations express the same NC PCDHs (panel (1)). See Table S1 for additional information
including N for each experiment.
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Fig. 2. Mosaic expression of Pcdh19 causes altered network brain activity and abnormal cell
sorting between PCDH19 positive and PCDH19 negative cells in the developing cortex. (A)
Representative 5 minute traces from ECoG recordings of +/+, +/β-Geo and β-Geo/β-Geo P42 mice.
(B) Quantification of SWD/hr of +/+, +/β-Geo and β-Geo/β-Geo P42 mice (****P < 0.0001, **P <
0.01, ns=not significant, student’s two-tailed, unpaired t test p values). (C) Quantification of mean
SWD duration of +/+, +/β-Geo and β-Geo/β-Geo P42 mice (****P < 0.0001, *P < 0.05, ns=not
significant, two-way ANOVA with Tukey’s multiple comparisons test). (D) Representative HA and
X-gal immunostaining of 14.5 dpc HA-FLAG/+ and HA-FLAG/β-Geo brains. Large blocks of HA-
positive and HA-negative cells are present in the ventricular zone (VZ) of the cortex which extended
into the intermediate zone (IZ) and cortical plate (CP) of HA-FLAG/β-Geo embryos (left). In contrast,
small patches of HA-negative cells were present along the ventricle of HA-FLAG/+ (indicated by
arrowheads) which did not extend into the intermediate zone (IZ) and cortical plate (CP) (right). (E)
Quantification of HA-negative regions in the cortex of 14.5 dpc HA-FLAG/β-Geo and HA-FLAG/+
and brains (**P < 0.01, unpaired t test). (F) Quantification HA-immunostaining intensity throughout
the cortex revealed a higher degree of variability in HA-FLAG/β-Geo compared with HA-FLAG/+
(*P < 0.05, unpaired t test). (G) Schematic describing CRISPR-Cas9-induced conversion of HA-
FLAG/β-Geo embryos to DEL/β-Geo. Two gRNAs targeting exon1 flanking sequences (red arrows)
were injected into HA-FLAG/β-Geo zygotes in combination with CAS9 protein. Zygotes were
transferred into pseudopregnant female mice and harvested 14 days post transfer. (H) HA and X-gal
immunostaining of HA-FLAG/β-Geo and DEL/β-Geo 14 day post transfer embryonic brains).
Normal redistribution of PCDH19 null cells was observed in the cortex of DEL/β-Geo embryos
indicating that preventing the disruption of PCDH19-dependent adhesion codes abolishes abnormal
cell sorting. (I) Quantification of X-gal staining intensity throughout the ventricle revealed a higher
degree of variability in HA-FLAG/β-Geo brains compared to HA-FLAG/+ brains (**P < 0.01,
unpaired t test). Also see Table S1.
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Fig. 3. Early cortical progenitor cells are acutely sensitive to mosaic expression of Pcdh19. (A)
Representative images of HA and SOX3 (neuroprogenitor marker) co-immunostaining of 9.5dpc and
10.5dpc HA-FLAG/+ and HA-FLAG/β-Geo heads (N=3 of each genotype). Abnormal cell sorting is
not apparent at 9.5dpc (right) but one day later the segregation of PCDH19-postive and PCDH19-
null cells has occurred (left). (B) HA immunostaining of P7 HA-FLAG/+ and HA-FLAG/β-Geo
brains (N=3 of each genotype) highlights the persistence of abnormal cell sorting in the postnatal
cortex.
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Fig. 4. Mosaic expression of PCDH19 results in variable cortical folding abnormalities in
PCDH19-GCE patients. Patient A: A focal area of cortical thickening in the left mid frontal lobe
(open arrow) and mild cortical thickening and blurring of the grey/white interface in the left posterior
temporal lobe was observed in axial T1 weighted MRI images (closed arrow). Patient B: Axial T1
weighted MRI images show an unusual stellate configuration to the right central sulcus with loss of
cortical clarity (open arrow) and a focal retraction/puckered appearance of right lateral frontal cortex
with complex sulcation and deep cortical thickening suggesting a likely bottom of the sulcus
dysplasia (closed arrow). A focal area of suspected cortical dysplasia was identified in the left mid
frontal region in addition to cortical thickening (arrowhead). Patient C: Axial and sagittal images
show an unusual complex sulcal arrangement present in the right frontal lobe with 2 parallel sulci
forming an ovoid ring configuration (closed arrows). Patient D: Contiguous coronal T1 images reveal
cortical thickening with blurring of the grey/white junction which is highly suggestive of a left
cortical dysplasia (closed arrows).
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Supplementary Materials for
Abnormal cell sorting is associated with the unique X-linked inheritance of
PCDH19 Epilepsy
Daniel T. Pederick1, 2, Kay L. Richards3, Sandra G. Piltz1, 2, Simone A. Mandelstam4, 5, 6,
Russell C. Dale, Ingrid E. Scheffer3, 7, Jozef Gecz1, 2, 8, 9, Steven Petrou3, James N. Hughes1,
2 and Paul Q. Thomas1, 2, 9*
This PDF file includes:
Materials and Methods
Fig. S1 to S12
Table S1 and S2
Complete References
Materials and methods:
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Plasmids
Open reading frames (ORF) were purchased from either Vector Builder (PCDH10 and
PCDH17) or Promega (PCDH19). Tagged ORFs were generated by PCR and point
mutations were created using overlap extension PCR. All ORFs were cloned into GFP or
Mcherry containing expression plasmids using restriction enzymes. Primer sequences used
for PCR amplifications will be provided upon request.
Co-immunoprecipitation and western blotting
K562 cells (human leukemia cell line, ATCC CCL243) were transfected with 5µg of each
plasmid using the Neon Transfection System (Invitrogen) and harvested 48 hours later.
Transfected cells were homogenized in IP lysis buffer (25mM Tris-HCl (pH7.4), 150 mM
NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol) in addition to cOmplete mini EDTA-free
Protease Inhibitor Cocktail (Roche) and the PhosSTOP Phosphatase Inhibitor Cocktail
(Roche). Cells were incubated in lysis buffer for 30 minutes at 4˚C, followed by brief
sonication and another 30 minutes at 4˚C. The supernatant was collected by centrifugation
at 13,200 rpm for 30 minutes at 4˚C. 1uL of mouse anti-FLAG (Sigma, F3165), was added
to each supernatant and incubated overnight at 4˚C. 30µL of Dynabeads™ Protein G
(Invitrogen) was added to each sample and incubated for 2 hours at 4˚C. Beads were washed
3x in IPP150 wash buffer (150mM NaCl, 10mM Tris/HCl, 0.1% Igepal (Sigma) and 5%
glycerol) and resuspended in 2X SDS load buffer. Lysates were separated on Invitrogen
Bolt™ precast 4-12% polyacrylamide gels and transferred to PVDF membrane before being
blotted. Antibodies and their corresponding dilutions were: mouse anti-FLAG 1:5000
(Sigma, F3165) and rabbit anti MYC (Cell Signalling Technology #2272S) Membranes
were blocked in 5% BSA + 5% skim milk in tris-buffered saline + 0.1% Tween20 (TBST)
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and antibodies were incubated with the membrane in 5% BSA in TBST at 4°C overnight.
Membranes were developed using Bio-Rad Clarity Western ECL substrate and imaged using
a Bio-Rad ChemiDoc.
K562 Aggregation Assay
2x106 K562 (human leukaemia cell line, ATCC CCL243) cells were nucleofected with 10µg
of plasmid DNA using the Neon Transfection System (Invitrogen) and incubated for 24
hours. Cells were harvested, treated with DNase (1mg/mL) and passed through a 40µm cell
strainer (Falcon) to obtain a single cell suspension. 2x105 cells were added to each well of a
12 well tray and allowed to aggregate for 2-4 hours on a nutator at 37˚C/5% CO2. Aggregated
cells were imaged using a Nikon Eclipse Ti microscope. To quantify aggregate size images
were exported to ImageJ where they were subjected to an equal threshold transformation and
aggregate size was assessed using the “analyse particle” function. Raw data was transferred
to GraphPad Prism 7 where a Kruskal-Wallis test was performed.
Cytospin immunofluorescence
K562 cell aggregates were fixed in 2% PFA for 2 hours at RT, washed in PBS and
resuspended in 30% sucrose. Aggregate suspensions were then adhered to superfrost slides
(ThermoFisher) using a Shandon Cytospin 3 (1000rpm, 10 minutes). Immunofluorescence
was performed by blocking with 10% foetal calf serum for 1 hour and then incubating with
mouse anti-FLAG (Sigma, F3165) 1/1000 overnight at 4˚C and then secondary antibody for
2 hours at room temperature (donkey anti-mouse 488 (Jackson ImmunoResearch)). Images
were acquired on a Leica SP5 Confocal microscope.
K562 Mixing Assay
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2x106 K562 cells were nucleofected with 5µg of each plasmid DNA (10µg total for co-
nucleofections) and incubated for 24 hours. Cells were harvested, treated with DNase
(1mg/mL) and passed through a 40µm cell strainer (Falcon) to obtain a single cell
suspension. 1x105 cells from each sample were pooled and added to each well of a 12 well
tray and allowed to aggregate for 3 hours on a nutator at 37˚C/5% CO2. Aggregated cells
were imaged using a Nikon Eclipse Ti microscope. Each condition was performed in
biological triplicate with four technical repeats. Pearson’s correlation coefficient was
calculated using NIS Elements Advanced Research Software (Nikon). Raw data was
transferred to GraphPad Prism 7 where a one way ANOVA test was performed.
Electrocorticogram (ECoG) recordings
ECoG recordings were obtained from 6-week-old female mice for wild type (WT),
heterozygote (Het) and knock-out (KO) animals in DBA/2J background (>N9). Surgery for
ECoG electrode placement was performed as described previously (36). In brief, mice were
anesthetized with 2-4% isoflurane and 2 epidural silver “ball” electrodes implanted on each
hemisphere above the somatosensory cortices using the following co-ordinates with respect
to bregma: (ML + 3 mm and AP -0.7 mm). A ground electrode was positioned 2.5 mm rostral
and 1 mm lateral from bregma. Mice were allowed to recover for at least 72 hours post-
surgery. Continuous ECoGs were then recorded in freely moving animals for 3-hour epochs
during daylight hours following a standard 30-minute habituation period; a minimum of 9
hours recording was obtained for data analysis. Signals were band pass filtered at 0.1 to 200
Hz and sampled at 1 kHz using Powerlab 16/30 (AD Instruments Pty. Ltd., Sydney, NSW,
Australia). In a subset of mice simultaneous video/ECoG recordings were also obtained to
correlate behaviour. The spike wave discharge (SWD) signature of +/+, +/β-Geo and β-
Geo/ β-Geo mice on a DBA/2J background was 5-8Hz as previously reported for this genetic
background (37, 38). 5 Quantification of spike wave discharges (SWD) was done for each
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genotype based on rhythmic biphasic spikes with voltage at least two-fold higher than mean
background. Measurements for SWD duration were made for a total of 350 events, defined
as time from the first spike peak to final peak with a minimum of one-second duration.
Generation and Genotyping of Pcdh19HA-FLAG Mice
CRISPR gRNA was designed to target near the stop codon of PCDH19 (5’-
TATCGTTCTCTAAAGCCATC-3’) using CRISPR Design tool (crispr.mit.edu) and
generated in house by cloning 20 nt oligos into pX330-U6-Chimeric_BB-CBh-hSpCas9,
which was a gift from Feng Zhang (Addgene plasmid # 42230) according to the protocol
described in (39). The ssDNA HA-FLAG oligo was designed to insert HA-FLAG 5’ of the
Pcdh19 stop codon with 60bp of homology either side (5’-
CGGAAGGATGGTCGTGACAAGGAATCTCCTAGCGTGAAGCGTCTGAAGGATAT
CGTTCTCTACCCATACGATGTTCCAGATTACGCTGACTACAAAGACGATGACG
ACAAGTAAAGCCATCTGGGTTCGAGGAAGAGAGAACAGAAACCACACTGGCT
AGTGAAGATGTAGCAG-3’) (Integrated DNA Technologies (IDT)). Cas9 mRNA was
generated by IVT (mMessage) from pCMV/T7-hCas9 (Toolgen) digested with XhoI. The
gRNA (50ng/µL), Cas9 mRNA (100ng/µL) and ssDNA HA-FLAG donor oligo (100ng/µL)
(IDT) were injected into C57BL/6N zygotes, transferred to pseudopregnant recipients and
allowed to develop to term. Founder pups were screened for HA-FLAG insertions by PCR
across targeted region (F 5’-TAGCGTGAAGCGTCTGAAGG-3’, R 5’-
CAGGCAGTAGGGGTGTTCAG-3’) and run on an agarose gel. A larger product of 281bp
represented the HA-FLAG allele, while a smaller 230bp product represented the wild type
allele. Positive samples were Sanger sequenced to verify the correct insertion of the HA-
FLAG sequence. Routine genotyping was performed by agarose gel electrophoresis in which
PCR products generated with the above primers were separated. The gender of embryos was
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identified by PCR amplification of SRY (F 5’-CAGTTTCATGACCACCACCA-3’ and R
5’-CATGAGACTGCCAACCACAG-3’) and the gender of postnatal mice was assessed by
analysis of external genitalia. All animal work was conducted following approval by The
University of Adelaide Animal Ethics Committee (approval number S-2013-187, S-2014-
068, S-2014-199A) in accordance with the Australian code for the care and use of animals
for scientific purposes.
Protein Extraction and western blotting
Hippocampi for protein extraction were minced in extraction buffer (50mM Tris, 150mM
NaCl, 1% NP40 (Roche) and 1% Triton X-100 (Sigma)) and incubated at 4˚C for 30 minutes.
Lysates were separated on Invitrogen Bolt™ precast 4-12% polyacrylamide gels and
transferred to PVDF membrane before being blotted. Antibodies and their corresponding
dilutions were: mouse anti-PCDH19 1:200 (Abcam, ab57510), mouse anti-FLAG 1:5000
(Sigma, F3165), mouse anti-HA 1:1000 (Cell Signalling Technology #2367) and rabbit anti-
β-ACTIN 1:1000 (Cell Signalling Technology, #4697). Membranes were blocked in 5%
BSA + 5% skim milk in tris-buffered saline + 0.1% Tween20 (TBST) and antibodies were
incubated with the membrane in 5% BSA in TBST at 4°C overnight. Membranes were
developed using Bio-Rad Clarity Western ECL substrate and imaged using a Bio-Rad
ChemiDoc.
Immunofluorescence
8.5dpc, 9.5 dpc and 10.dpc whole embryos were dissected and fixed for 2-6 hours in 4%
paraformaldehyde (PFA) at 4˚C. To collect 13.5dpc, 14.5dpc and 15.5dpc embryonic brains
pregnant dams were cardiacally perfused with 4% PFA. Embryonic brains were dissected
and post fixed in 4% PFA at 4˚C for 4-6 hours. To collect P7 brains mice were cardiacally
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perfused with 4% PFA, brains dissected and post fixed in 4% PFA at 4˚C for 4-6 hours.
Embryos and brains were then cryoprotected in 30% sucrose and frozen in OCT embedding
medium. 16µm sections were cut using a Leica CM1900 cryostat. Sections were blocked
with 0.3% TritonX-100 and 10% horse serum in PBS for 1 hour at room temperature.
Sections were then incubated overnight with rabbit anti-HA 1:300 (Cell Signalling
Technology, #3724), goat anti-SOX3 1:300 (R&D Systems, AF2569), rat anti-CTIP2 1:400
(Abcam, ab18465) and rabbit anti-CUX1 1:400 (Santa Cruz, sc-13024) at 4˚C and then
secondary antibody for 2 hours at room temperature (donkey anti-rabbit TxRed (Life
Tehcnologies), donkey anti-goat 488 (Life Technologies), donkey anti-guinea pig 488
(Jackson ImmunoResearch) and goat anti-rat 488 (Jackson ImmunoResearch). Images were
acquired on a Nikon Eclipse Ti microscope false coloured using Adobe Photoshop.
X-Gal staining
14.5dpc brains were collected as described above. Staining was performed as previously
described (20). Images were acquired on a Nikon Eclipse Ti microscope and false coloured
using Adobe Photoshop.
X-gal and immunofluorescence co-staining
X-gal staining was performed then followed by immunofluorescence staining as described
above. Images were acquired on a Nikon Eclipse Ti microscope and false coloured using
Adobe Photoshop.
Quantification of HA negative areas
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Images from HA-FLAG/β-Geo transformed until HA-negative regions in HA-FLAG/β-Geo
cortices became blank pixels. Equal thresholding was performed on HA-FLAG/+ images.
Regions of interest were drawn around the cortex of all images and the percent of HA-
negative area (blank pixels) was assessed. Raw data was transferred to GraphPad Prism 7
where a Student’s T-test was performed.
Quantification of HA immunostaining intensity variability
21 regions of interest (50µm x 50µm) were placed throughout the cortex and intensity
measured using NIS Elements Advance Research Software (Nikon). Raw data was
transferred to GraphPad Prism 7 where the coefficient of variation was calculated.
Generation of DEL/β-Geo embryos from HA-FLAG/β-Geo zygotes
CRISPR gRNAs were designed to target immediately after the Pcdh19 ATG and the 3’ end
of exon 1 (5’-CGGGACGGTGATCGCTAACG-3’) and (5’-
AGATCCGGACCTACAATTGC) respectively and generated as above. The gRNA
(25ng/µL each) and CAS9 protein (50ng/µL) were combined and incubated for 10 minutes
on ice before being injected into Pcdh19HA-FLAG/β-Geo zygotes which were then transferred to
pseudopregnant recipients. 14 days post transfer embryonic brains were collected and fixed
for 4-6 hours in 4% PFA at 4˚C.
Embryos were screened for the presence of HA-FLAG alleles by PCR as described above to
confirm Pcdh19HA-FLAG/β-Geo identity. Pcdh19HA-FLAG/β-Geo embryos were then screened for
large deletions of exon1 by PCR ( F 5’-GCAATCTCAGCATTGGAGCC-3’) (R 5’-
GGGGTTTTTAGACCGACCGA-3’). Large deletions of exon 1 were confirmed via Sanger
sequencing. Pcdh19HA-FLAG/β-Geo embryos positive for large deletions were then processed
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for HA and X-Gal staining as described above, along with Pcdh19HA-FLAG/β-Geo embryos to
serve as controls.
Quantification of X-gal staining intensity variability
14 regions of interest (50µm x 50µm) were placed throughout the ventricular zone of the
cortex and intensity measured using NIS Elements Advance Research Software (Nikon).
Raw data was transferred to GraphPad Prism 7 where the coefficient of variation was
calculated.
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Fig. S1. PCDH19-GCE missense mutations lack adhesive function. (A) K562 cells transiently
expressing PCDH19 form aggregates whereas cells K562 cells expressing PCDH19-GCE mutations
do not. (B) Quantification of aggregate particle size confirms the lack of adhesive function in
PCDH19-GCE missense mutations. (n=2 biological repeats with 10 technical replicates, **, p<0.01,
****, p<0.0001). (B) FLAG immunohistochemistry of K562 cells, white arrows indicate enrichment
of PCDH19 at cell-cell interfaces (n=2 biological repeats with 4 technical replicates).
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Fig. S2. +/β-geo mice display increased SWD frequency, duration and epileptiform events
during sleep. (A) Representative trace showing multiple SWDs highlighting the increased frequency
of SWDs in +/β-Geo mice. (B) Single representative SWDs highlighting the increased duration of
SWDs in +/β-Geo mice.
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Fig. S3. Generation of Pcdh19HA-FLAG mice using CRISPR-Cas9 editing reveals PCDH19
expression in the developing and postnatal brain. (A) Pcdh19HA-FLAG targeting strategy showing
the HA-FLAG epitope (green), gRNA (red), PAM site (orange) and endogenous STOP codon
(underlined). (B) PCR amplification was used to identify the insertion of the HA-FLAG epitope tag.
(C) Protein extracts from P14 hippocampus were immunoblotted with PCDH19, FLAG, HA and β-
ACTIN antibodies. (D) HA-immunostaining of 14.5 dpc Pcdh19+/Y and Pcdh19HA-FLAG/Y (n=3). (E)
HA immunostaining of P7 Pcdh19HA-FLAG brains and co-staining with layer markers CTIP2 (layer
Vb) and CUX1 (layer II-IV) (n=3), reveals PCDH19 expression predominantly in layers I and V.
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Fig. S4. PCDH19 has widespread expression throughout brain development. HA-
immunostaining of HA-FLAG/Y embryonic brains revealed PCDH19 expression in the cortex,
hippocampus, ganglionic eminence and sub-cortical regions. Representative images of N=3 for each
time point.
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Fig. S5. Raw data used to quantify the variation seen between HA-FLAG/β-Geo animals. (A)
Box and whisker plot demonstrating range of HA immunostaining intensity values for each
independent animal. (B) Box and whisker plot demonstrating range of X-gal staining intensity values
for each independent animal.
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Fig. S6. Abnormal cell sorting patterns in HA-FLAG/β-Geo brains is variable. (A) HA
immunostaining of HA-FLAG/+ brains at 13.5, 14.5 and 15.5 dpc. (B) HA immunostaining of HA-
FLAG/β-Geo brains at 13.5, 14.5 and 15.5 dpc. (C) X-Gal staining of HA-FLAG/β-Geo brains from
(B) merged with HA images.
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Fig. S7. PCDH19 is not present at cell-cell interfaces between WT PCDH19 and null PCDH19
radial glial cells. (A) HA and Phalloidin staining of the ventricular lining of 10.5 dpc cortex (N=3
of each genotype). PCDH19 is present at all cell-cell boundaries in HA-FLAG/Y brains (left,
arrowheads) and at boundaries between HA-positive and HA-negative cells in HA-FLAG/+ brains
(middle, arrowheads). In contrast, PCDH19 is not present at boundaries between HA-positive and
HA-negative cells providing further evidence of its homotypic nature in vivo (right, arrowheads).
(B) Schematic representation of PCDH19 location (green lines) overlapping with Phalloidin positive
cell-cell interfaces (black lines).
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Fig. S8. Cell segregation in the developing cortex is due to the redistribution of cell bodies. (A)
SOX3 immunostaining of Pcdh19+/+ : Sox3+/- 13.5 dpc cortex shows normal X-inactivation (N=3).
(B) SOX3 staining of Pcdh19+/β-Geo :Sox3+/- in 13.5 dpc cortex shows abnormal cell sorting of SOX3
+ve and SOX3 –ve neural progenitor cells (N=3). SOX3 null mice were generated by CRISPR-Cas9
mediated deletion of the whole ORF (unpublished data).
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Fig. S9. PCDH19 is first expressed at 9.5dpc in the developing cortex. (A) At 8.5dpc PCDH19
expression is present in the surface ectoderm but no in the SOX3 +ve neuroepithelial cells (N=3) (B)
PCDH19 expression is present in the developing cortex at 9.5dpc and 10.5 dpc (N=3 of each time
point).
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Fig. S10. PCDH19 expression is enriched in the human cortex throughout embryonic
development. (A) RNA-seq data from the Allen Brain Atlas highlighting the differential expression
of PCDH19 in brain regions throughout development (26). (B) Data from RNA-Seq of human
individual tissues and mixture of 16 tissues (Illumina Body Map) was retrieved from EMBL-EBI
ArrayExpress database under accession number E-MTAB-513
(http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-513/) on 09/01/2016 highlighting the
differential expression of PCDH19 across different regions of the adult human. (White boxes
represent no data available).
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Fig. S11. Pedigrees of PCDH19-GCE patients which present with cortical folding
abnormalities. (A) Acquired mutation through paternal inheritance (21). (B) Mutation was de novo
(unpublished data). (C) Mutation was de novo (unpublished data). (D) Acquired mutation through
paternal inheritance (unpublished data).
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Fig. S12. Mosaic expression but not the complete loss of PCDH19 leads to incompatible
adhesion codes. (A) Wild type cells in normal individuals have a complementary adhesion code
(AC) which contains PCDH19, allowing interaction between cells. (B) Mosaic expression of
PCDH19 leads to a mixture of cells that contain different adhesion codes. This causes wild type (AC)
and null (AC-19) cells to have different adhesion codes and interact with cells that have a
complementary adhesion code. (C) The complete loss of PCDH19 uniformly removes PCDH19 from
all cells and therefore does not affect complementary adhesion codes.
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Table S1. Raw data from quantitative analysis, Fig. 1-2.
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Table S1. Brief clinical description of PCDH19-GCE patients which have cortical folding
abnormalities. FIAS, focal impaired awareness seizure; GTCS, generalised tonic-clonic seizure;
FBTC, focal bilateral tonic-clonic seizure; T, tonic; FT, focal tonic; H, hemiclonic; Ab, absence; MJ,
myoclonic jerks; At, atonic; m, months; y, years; F, female; M, male; GSW, generalised spike-wave;
AED, anti-epileptic drug; PSW, polyspike-wave; w, weeks; SE, status epilepticus; DD,
developmental delay; VEM, video EEG monitoring; CSE, convulsive status epilepticus; ID,
intellectual disability; pat inh, paternally inherited; VNS, nagal nerve stimulator; LEV, levetiracetam;
VPA, sodium valproate; LTG, lamotrigine; PB, phenobarbitone; PHT, phenytoin; CBZ,
carbamazepine; CZP, clonazepam; CLB, clobazam; TPM, topiramate; KD, ketogenic diet; OCD,
obsessive compulsive disorder. *exacerbated seizures. Current medications underlined.
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CHAPTER 4
General Discussion
125
CHAPTER 4: General Discussion
126
Together the data presented from Chapters 2 and 3 indicate that loss of Pcdh19 does not
overly affect mouse brain development but when expressed in a mosaic fashion causes
abnormal cell sorting between Pcdh19 WT and Pcdh19 null cells. Furthermore, we show
that abnormal cell sorting in the developing cortex correlates with altered network brain
activity consistent with changes that can underlie seizures which is not observed in mice
completely lacking Pcdh19. We also provide evidence supporting the combinatorial activity
of NC PCDHs contributing to specific cell-cell interactions, which when disrupted leads to
abnormal cell sorting. Overall we provide a long sought after cellular mechanism for the
unique X-linked inheritance pattern of PCDH19-GCE. However, the examination of results
from Chapters 2 and 3 together raise additional discussion points which are addressed below.
In Chapter 2 we reported no gross morphological abnormalities in Pcdh19+/β-Geo and
Pcdh19β-Geo/β-Geo brains and identified no spontaneous seizures in Pcdh19+/β-Geo mice. This
may be due to the inherent difficulty of observing seizures in neonatal mice (the equivalent
age where clusters of seizures present in PCDH19-GCE patients) or genetic differences
between mice and humans. In fact, attempts to model epilepsy in mice do not always
genetically match the corresponding human disorder. For example, heterozygous TSC1/2
mutations in humans lead to Tuberous Sclerosis and seizures but an epilepsy phenotype is
not observed in mice with heterozygous Tsc1/2 loss of function mutations (European
Chromosome 16 Tuberous Sclerosis Consortium, 1993; Fryer et al., 1987; Kobayashi et al.,
2001; Onda et al., 1999). Similarly, heterozygous mutations in PRRT2 cause a range of
epileptic and movement disorders that has recently been modelled using a Prrt2 null mouse
(Michetti et al., 2017). Phenotypes representing epilepsy and movement disorders were only
reported in Prrt2 homozygous mice and no pathogenic phenotype was observed in Prrt2
heterozygous null mice. Despite the lack of seizures in Pcdh19+/β-Geo mice, it is important to
CHAPTER 4: General Discussion
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note that ECoG analysis identified an epileptic like phenotype. Therefore, the PCDH19-
GCE mouse model does follow the human inheritance pattern since Pcdh19+/β-Geo mice
display epileptiform brain activity and Pcdh19β-Geo/β-Geo mice do not. We therefore conclude
that heterozygous mice are an appropriate informative model to investigate the cellular
mechanisms that underlie the disease. It appears likely that inherent differences in human
and mouse brain development are the reason for the presentation of different phenotypes.
We also show that the loss of Pcdh19 leads to a subtle increase in the migration potential of
neurons in vitro. This was not seen in vivo as there were no ectopically positioned Pcdh19
null neurons present in both the developing and adult brain. This was further supported by
our findings in Chapter 3, where Pcdh19 WT and Pcdh19 null cells were both correctly
positioned when simultaneously identified within the same brain. We therefore believe it is
unlikely that the subtle migration defect seen in cultured Pcdh19 null neurons contributes to
pathogenesis as we confirmed mice completely lacking Pcdh19 have normal brain activity.
Instead the slightly increased migration potential may highlight another endogenous role of
Pcdh19 that does not contribute to PCDH19-GCE.
The abnormal cell sorting of neuroepithelial progenitors and radial glia raises the intriguing
possibility that the entire mature cortex segregates according to their developmental
expression of Pcdh19. If the entire cortex is subjected to rearrangement due to mosaic
Pcdh19 expression, it is possible the epileptiform brain activity observed in adult Pcdh19+/β-
Geo is not only caused by defective connectivity between Pcdh19 WT and Pcdh19 null
neurons but also contributed to by abnormal connections and spatial arrangement of other
neurons. If the entire cortex was to undergo segregation it is likely this would result in
abnormal organisation of functional cortical columns. Interestingly, cortical column
organisation has been reported in gyrencephalic (folded) brains such as apes, monkeys and
ferrets but there is little evidence supporting cortical column organisation in the
CHAPTER 4: General Discussion
128
lissencephalic (smooth) brains of mice and rats (Chow et al., 1971; Shaw et al., 1975; Tiao
and Blakemore, 1976). If abnormal cell sorting does occur throughout the entire cortex this
neuronal architecture difference between humans and mice may contribute to the more
severe phenotype seen in PCDH19-GCE patients. Furthermore, the identification of
abnormal cortical sulcation in multiple PCDH19-GCE patients, suggests that aberrant cell
sorting may generate different morphological phenotypes in gyrencephalic brains compared
to lissencephalic brains. The cortical folding of human brains is largely caused by variable
proliferation of basal radial glia, a cell type which is not present in the lissencephalic brain
of mice. Proliferation of basal radial glia cause “wedges” of cell dense areas, which
ultimately cause the brain to fold. It is possible that if segregation occurred within the basal
radial glial cells in the developing human brain then abnormal folding may occur due to
irregular formation of “wedges”. These hypothesises could be investigated by using
CRISPR-Cas9 technology to generate a Pcdh19+/- ferret (Kou et al., 2015), a gyrencephalic
animal model, allowing any roles of PCDH19 specific to folded brains to be examined.
X-Gal staining of Pcdh19+/β-Geo mice in Chapter 2 revealed “patchy” staining of Pcdh19 null
cells which we attributed to normal X-inactivation. Due to the clonal and radial nature of
excitatory neuron migration in the cortex we predicted X-inactivation would manifest as
“columns” of either Pcdh19 WT or Pcdh19 null cells. This was in agreement with previous
studies that utilised an X-linked β-galactosidase reporter where predominant columns of X-
Gal staining were present in the mouse cortex (Tan et al., 1995). At the time this research
was undertaken the normal X-inactivation pattern of neurons in the cortex was not well
understood. More recently, experiments have been performed to conduct a detailed analysis
of X-inactivation in the mouse brain, by labelling both X-chromosomes with either GFP or
tdTomato (Wu et al., 2014). It was reported that although there were regions in the cortex
CHAPTER 4: General Discussion
129
predominantly originating from one X-chromosome there was always a degree of
intermingling of both GFP and tdTomato positive cells. This agrees with the HA staining of
control Pcdh19HA-FLAG/+ brains which showed that normal X-inactivation leads to grossly
even distribution of PCDH19. Furthermore, these results highlight the significance of our
finding that normal cell distribution is dramatically affected by mosaic expression of
Pcdh19. The fact that we misinterpreted the “patchy” staining in heterozygous mice
highlights the importance of controls. In our case it was not until we had generated a HA
tagged mouse and stained HA/+ control mice that we were able to properly interpret the
“patchy” staining in heterozygous mice as an abnormal cell sorting phenotype.
Using a combination of in vitro and in vivo techniques we have shown that the abnormal cell
sorting is due to disruption of PCDH19 dependent adhesion interfaces between
neuroprogenitors cells at 10.5 dpc. During cortical development neuroprogenitors located in
the ventricular zone predominantly undergo radial migration. We envisage two potential
cellular consequences downstream of incompatible adhesion codes that could lead to
segregation of neuroprogenitors; 1) the incompatibility between cells prevents lateral mixing
(passive segregation); or 2) the incompatibility between cells promotes active sorting of
neuroprogenitors. Our results suggest that the cells undergo active sorting as we observe
small “blocks” only a few cells wide in 14.5-17.5 dpc brains. If lateral mixing was prevented,
we would expect to see a minimum “block” size that would represent proliferation of a single
“trapped “cell at 10.5 dpc (32 cells at 14.5 dpc with an assumed cell cycle of 12 hours). One
caveat of our experiment is the two-dimensional analysis. It is possible, the small blocks we
see at later stages are because we have “shaved” the edges of larger blocks. Analysis of the
segregation pattern in three-dimensions would clarify this mechanism.
CHAPTER 4: General Discussion
130
We show that PCDH19 and other members of the NC PCDH family can function in
combinatorial activity which dictates cell-cell interaction specificities. This is similar to the
cooperative activity of clustered PCDHs which have been proposed to form lattice like
structures that act to differentiate between self and non-self neuronal processes (Rubinstein
et al., 2015). Structural studies have further elucidated this mechanism showing that
clustered PCDHs form tetramers that are maintained by trans homodimers through EC
domains 1-4 and cis heterodimers through EC6 (Goodman et al., 2016; Nicoludis et al.,
2015; Rubinstein et al., 2015). The mechanism by which NC PCDHs act in combination is
still to be determined and could potentially be similar to the lattice like structures formed by
clustered PCDHs. This is supported by the recent structure analysis of zebrafish PCDH19
highlighting its similarity to clustered PCDHs (Cooper et al., 2016). In addition, using co-
immunoprecipitation we showed that PCDH19 can interact with both PCDH17 and PCDH10
when expressed in the same cell, supporting the ability of NC PCDHs to form cis
heterodimers. In the future disease causing mutations positioned in domains important for
trans (EC1-4) and cis (EC6) interactions could be assessed to determine how they impact
the formation of trans and cis interactions. Cooper et al., 2016 showed that missense
mutations within EC1-4 are positioned in domains crucial for homotypic trans interactions
and we would predict that missense mutations within EC6 will affect cis heterodimeric
interactions.
Rubinstein et al., 2015 modelled the impact a single PCDH isoform mismatch has on the
size of lattice structure between cells. They showed that a single mismatch between the
PCDH profiles of two adjacent cells led to a dramatic decrease in lattice size, which they
attributed to cause cell sorting in their in vitro experiments. We used the same modelling
approach to determine if having one less PCDH between cells, as is the case in vivo using
Pcdh19HA-FLAG/β-Geo mice, would result in the same dramatic decrease in lattice size. We show
CHAPTER 4: General Discussion
131
that the lattice size between two cells where one cell has one less PCDH (N:N-1) modelling
Pcdh19HA-FLAG/β-Geo mice, is dramatically reduced compared to a perfect match of PCDHs
between two cells (N:N) (Figure 1-4). Furthermore, if both cells have the same PCDH
removed (N-1:N-1), modelling Pcdh19β-Geo/β-Geo, mice the lattice size returns to almost
normal levels, which agrees with the absence of a phenotype observed in Pcdh19β-Geo/β-Geo
(Figure 1-4). In agreement with this we showed that we could remove NC PCDH
incompatibility between PCDH19 WT and PCDH19 null cells by deletion of the remaining
functional Pcdh19 allele in Pcdh19HA-FLAG/β-Geo mice which lead to reversal of abnormal cell
sorting.
CHAPTER 4: General Discussion
132
Figure 1-4. The predicted size of adhesion complexes between PCDH19 WT and PCDH19 KO
cells is dramatically reduced. Mathematical modelling predicts the size of adhesion complexes is
dramatically reduced between PCDH19 WT and PCDH19 KO cells (green line) when compared to
adhesion complexes between PCDH19 WT and PCDH19 WT (red line) or PCDH19 KO and
PCDH19 KO cells (green line). Modelling was performed with 100 isomers of each PCDH molecule.
(Performed by Ian Dodd).
CHAPTER 4: General Discussion
133
NC PCDHs could act in combination by determining the adhesive strength of cells. Cell
sorting could then occur as cells with higher adhesion potential will cluster and the cells with
lower adhesion potential will be excluded but still form their own cluster. We believe this
mechanism is unlikely for multiple reasons; 1) cells in the developing cortex express many
adhesion molecules, therefore it is unlikely the removal of just PCDH19 (a relatively weak
adhesion molecule) would lead to a dramatic decrease in their adhesion potential; 2) the
absence of a neurological phenotype in Pcdh19β-Geo/β-Geo mice suggests that cells completely
lacking PCDH19 are largely normal and 3) in vitro experiments where two populations of
cells expressed the same number but different NC PCDH molecules displayed cell
segregation – we would expect many of these cells to have similar overall adhesion strength.
134
CHAPTER 5
Future Directions
135
CHAPTER 5: Future Directions
136
Here we have shown that abnormal cell sorting underlies the unique X-linked inheritance
pattern of PCDH19-GCE. This provides a platform to investigate how abnormal cell sorting
leads to seizures, intellectual disability and autism spectrum disorder. A series of
experiments is described below to identify additional neurological processes which are
disrupted due to the mosaic expression of Pcdh19 which may contribute to pathology.
5.1 Investigating Connectivity between PCDH19 WT and PCDH19 null
neurons
Given the expression of PCDH19 at the synapse (Chapter 2) and the known roles for δ2
PCDHs in axon guidance and synaptogenesis (Hayashi et al., 2014; Uemura et al., 2007) we
envisage that synaptic connections between PCDH19 WT and PCDH19 mutant cells will be
disrupted in PCDH19 heterozygous females contributing to disease. Furthermore, the
morphology of neurons positioned at boundaries between PCDH19 WT and PCDH19 KO
cells may be affected. We hypothesise that abnormal cell sorting of PCDH19 WT and
PCDH19 KO cells alters neuronal connectivity through disruption of lateral connections
within the cortex. This can be investigated by generating a Pcdh19-P2A-CRE mouse
(Pcdh19Cre), permitting the expression of CRE in cells expressing functional PCDH19.
Pcdh19Cre mice could then be used in two ways to investigate morphology and arborisation
of PCDH19 neurons. The first approach would cross Pcdh19Cre mice to commercially
available Brainbow 3.2 mice (Cai et al., 2013), which generate multi-coloured neurons in
response to Cre recombination which variably activates three membrane-bound fluorophores
to create a range of colours. This will allow individual PCDH19 expressing neurons to be
traced even in large groups of PCDH19 positive cells allowing us to test the hypothesis that
neuronal processes will also be segregated in addition to cell bodies. Secondly in utero
electroporation of mouse embryos with a loxP-membrane_tdTomato-loxP-membrane_GFP
CHAPTER 5: Future Directions
137
construct (pCA-mT/mG – Addgene plasmid #26123) will result in mGFP staining only in
Pcdh19-Cre positive cells while Pcdh19-Cre negative cells will express mtdTomato.
Comparing Scholl analysis of mGFP neurons in Pcdh19Cre/+ and Pcdh19Cre/β-Geo brains will
assess any reduction in arborisation formed at the boundaries of PCDH19 WT and PCDH19
KO cells in the cortex.
To investigate functional synaptic connectivity between PCDH19 WT and PCDH19 KO
cells a modified rabies virus (EnvA-delta;G) (DeNardo et al., 2015) can be used to assess
the degree of monosynaptic transmission. This approach would also utilise the Pcdh19Cre
mice as the modified rabies virus is expressed in a Cre-dependent fashion, allowing only
Pcdh19-Cre cells to become competent for infection. Initially a cre-dependent helper AAV
virus will be injected into the cortex to express TVA-mCherry, which is essential for rabies
virus infection. 14 days later the rabies-GFP virus will be injected and brains analysed 5 days
later. This approach marks “starter” cells in yellow and “input” cells in green allowing robust
identification of cells which have received synaptic inputs. We would expect to see reduced
connectivity in Pcdh19Cre/β-Geo brains compared to Pcdh19Cre/+ brains. Furthermore, we
would expect that connectivity when present in Pcdh19Cre/β-Geo brains will be restricted to
cells within the same patch of Pcdh19-Cre cortex and will not propagate into PCDH19 null
zones. The same technique could also be applied in vitro on cultured primary neurons from
Pcdh19Cre/+ and Pcdh19Cre/β-Geo embryos.
CHAPTER 5: Future Directions
138
5.2 Is Interneuron Distribution Affected by Mosaic Pcdh19 Expression?
Inhibitory neurons (interneurons) are responsible for keeping neuronal circuits “in check”
and maintaining proper firing rates between excitatory neurons. It has been suggested that
altered inhibition due to interneuron dysfunction is an underlying cause of epilepsy (Bernard
et al., 2000; Holmes, 1997; Tasker and Dudek, 1991).
Cortical interneurons begin life in the ganglionic eminence and undergo a long range
tangential migration path which relies on a large number of molecular cues. Physical, genetic
or chemical disruptions in brain development can impair interneuron function resulting in
unstable neural circuits, evolving into seizures. Due to the relatively low number of
interneurons compared to excitatory neurons in the mammalian cortex (approximately 1:3
(Markram et al., 2004)) it is crucial that they are appropriately distributed to allow correct
network inhibition.
We have evidence that PCDH19 is expressed in both interneuron progenitors in the
ganglionic eminence and tangentially migrating interneurons in the marginal zone of the
developing cortex (Figure 1-5A). Furthermore, segregation of PCDH19-positive and
PCDH19-null interneurons is observed in both the ganglionic eminence and marginal zone
(Figure 1-5B and C). Therefore, we hypothesise that segregation of PCDH19-positive and
PCDH19-null interneuron precursors during tangential migration leads to abnormal
distribution of cortical interneurons in the mature brain. In the future it would be interesting
to test whether the final location of PCDH19-positive interneurons is segregated and whether
this segregation corresponds with the abnormal cell sorting pattern observed in excitatory
cells of the cortex. We feel there are two possible presentations of interneuron segregation;
interneurons will segregate according to PCDH19 expression in excitatory cells, such that
CHAPTER 5: Future Directions
139
PCDH19 expressing interneurons only populate PCDH19 expressing zones of the cortex
(Figure 2-5, left); alternatively, segregation established during migration of interneurons is
the determining factor in final distribution and not abnormal cell sorting in cortical excitatory
cells (Figure 2-5, right). To investigate this, we will utilise the Pcdh19Cre mouse described
above to specifically label PCDH19 expressing interneurons in a heterozygous brain and see
where they are finally positioned. We will specifically label PCDH19-Cre positive
interneurons by in utero electroporation of mT/mG plasmid into the ganglionic eminence of
Pcdh19Cre/β-Geo embryos. This technique will allow us to trace the lineage of mature
interneurons expressing PCDH19 throughout development as they will be marked by mGFP
due to Cre-recombination. Mature brains will be stained for HA to show the cortical cell
sorting pattern and GFP to reveal the location of PCDH19-Cre positive interneurons.
If PCDH19-positive interneurons are restricted to PCDH19-positive patches of cortex we
envisage a disturbance in the distribution of interneurons. We have evidence showing the X-
inactivation ratio in the cortex can be vastly different from the X-inactivation pattern in the
ganglionic eminence in the same hemisphere which could lead to a situation where there is
an insufficient number of PCDH19-positive interneurons or PCDH19-negative interneurons
to distribute amongst the appropriate patches in cortex. This would result in regions with
increased excitation due to lack of interneurons. Alternatively, if interneuron distribution is
unaffected it will result in PCDH19 WT interneurons being present in PCDH19 KO patches
of the cortex and vice versa. As we suggested above, synaptic connections between PCDH19
WT and PCDH19 KO neurons may be perturbed, therefore inhibitory synapses in these
regions may be disrupted, again leading to increased excitation.
CHAPTER 5: Future Directions
140
Figure 1-5. PCDH19 is expressed in tangentially migrating cortical interneurons which
undergo abnormal cell sorting. A) Co-immunostaining of 15.5 dpc HA-FLAG/Y brains reveals
PCDH19 expression in tangentially migrating cortical interneurons. B) HA and X-gal staining of
14.5 dpc HA-FLAG/β-Geo brains shows segregation of PCDH19 positive and PCDH19 null cells in
the ganglionic eminence. C) HA and X-gal staining of 16.5 dpc brains confirms abnormal cell sorting
of tangentially migrating interneurons in the marginal zone.
HA-FLAG/Y
HA-FLAG/β-Geo HA-FLAG/β-Geo
A
B C
CHAPTER 5: Future Directions
141
Figure 2-5. Schematic diagram describing different mechanisms that may lead to abnormal
interneuron distribution. It is proposed that the final position of interneurons in the mature cortex
is dictated by either their pre-segregation pattern (left) or selective homing to “PCDH19-like” regions
of excitatory cells (right).
Pre-segregated Pattern Selective Homing
PCDH19KO pyramidal progenitor
PCDH19KO interneuron progenitor
PCDH19HA-FLAG pyramidal progenitor
PCDH19HA-FLAG interneuron progenitor
CHAPTER 5: Future Directions
142
To complement the above experiments investigating the role of PCDH19 in the final
positioning of interneurons we could generate a floxed Pcdh19 allele (Pcdh19fl) which would
result in complete loss of PCDH19 in the presence of Cre. Pcdh19fl mice will be crossed
with Dlx-Cre mice which will lead to the deletion of Pcdh19 in all cortical interneurons. We
hypothesise that PCDH19 negative interneurons will fail to be positioned correctly in the
cortex leading to decreased local inhibition of excitatory neurons. Furthermore, Pcdh19fl
mice could be crossed with Emx1-Cre mice which will delete Pcdh19 from all excitatory
pyramidal neurons of the cortex. Again we would hypothesise that Pcdh19 positive
interneurons will fail to be positioned correctly in the cortex. These experiments will
investigate the importance of PCDH19 expression in both excitatory and inhibitory neurons
of the cortex and its role in determining the final position of cortical interneurons. They
could also highlight a role for PCDH19 in dictating specific cell-cell interactions not only
within the same cell type as we described in Chapter 3, but also between different types of
neurons.
CHAPTER 5: Future Directions
143
5.3 Can Epileptiform Brain Activity be Rescued after Abnormal Cell
Sorting has Occurred?
In Chapter 3 we show that deletion of PCDH19 in heterozygous female embryos reverses
abnormal cell sorting. This experiment was performed in zygotes meaning that Pcdh19 was
deleted before its initial expression in the developing brain at 9.5dpc and consequently
before abnormal cell sorting occurs. It would be interesting to investigate whether the
deletion of Pcdh19 after abnormal cell sorting has begun can rescue the phenotype.
Furthermore, if PCDH19 is important in forming homotypic interactions during
synaptogenesis, deletion of Pcdh19 in the postnatal brain may restore some normal neural
connectivity even though abnormal cell sorting occurred during development. We
hypothesise that abnormal cell sorting will only be rescued if Pcdh19 is deleted before
9.5dpc in the mouse brain, but deletion of Pcdh19 in the postnatal brain will rescue abnormal
synaptic connections that may occur between PCDH19 WT and PCDH19 KO cells. To test
this, Pcdh19fl mice could be crossed with an inducible global CRE mouse line where Cre is
activated in the presence of doxycycline (Dox). Dox will then be introduced to pregnant
female mice at different stages to determine whether deletion of Pcdh19 can rescue abnormal
cell sorting. Furthermore, Pcdh19fl/β-Geo adult mice could be subject to ECoG analysis to
assess their level of increased brain activity and then be administered with Dox followed by
another ECoG analysis. This will determine if deletion of Pcdh19 in the adult brain can
restore neuronal connections between PCDH19 WT and PCDH19 KO cells returning circuit
activity to normal.
The experiments outlined above will provide further insight into how abnormal cell sorting
ultimately leads to epileptiform brain activity in Pcdh19HA-FLAG/β-Geo mice. This information
will be critical in understanding the origin of seizures in PCDH19-GCE and the development
of much needed novel treatments.
CHAPTER 5: Future Directions
144
5.4 Investigating the Combinatorial Activity of Other NC PCDHs In Vivo
To support our data suggesting that NC PCDHs cooperate to dictate specific cell-cell
interactions in the developing brain, it would be interesting to investigate whether cell
sorting occurs with mosaic expression of other NC PCDH members. This experiment is
particularly challenging as all other PCDHs are autosomal genes, unlike PCDH19 which is
X-linked, and therefore undergoes X-inactivation generating a mosaic expression pattern.
One approach to perform this experiment would be to utilise Pcdh19Cre mice as a system
that can recombine DNA in a manner resembling X-inactivation. This would also require
the generation of a floxed gene of interest, for example Pcdh10. In order to label PCDH10
WT and PCDH10 KO cells the floxed allele would be generated using CRISPR-Cas9
technology in zygotes of mT/mG strain (Muzumdar et al., 2007). This mT/mG strain
operates in the same way as the above described plasmid where cells express membrane-
GFP in the presence of Cre, while cells are membrane-tdTomato positive in the absence of
Cre. Mice would be generated that are homozygous for both the mT/mG allele and the
floxed-PCDH10 allele. These mice could then be crossed with Pcdh19Cre/+ mice to generate
mosaic expression of PCDH10 WT and PCDH10 KO cells that are labelled with mtdTomato
and mGFP respectively, allowing easy identification of a cell sorting phenotype (Figure 3-
5).
Alternatively, PCDH10 KO mouse embryonic stem cells (mESC) that contain a GFP marker
could be generated and injected into mouse blastocysts. The localisation of PCDH10 KO
ESCs could be compared to that of PCDH10 WT ESCs in the developing cortex to
investigate a cell sorting phenotype. Both of the describe techniques could be performed
with multiple NC PCDHs to further strengthen a combinatorial role in the developing brain
CHAPTER 5: Future Directions
145
Figure 3-5. Experimental design to investigate the effect mosaic expression of PCDH10 has on
cortical cell sorting. Mice will be generated that display mosaic expression of PCDH10 due to Cre
recombination driven by the X-linked Pcdh19Cre allele. We hypothesis that abnormal cell sorting will
occur in animals with mosaic PCDH10 expression but normal cell sorting will occur in animals that
are either complete KO or HET for PCDH10.
CHAPTER 5: Future Directions
146
5.5 Concluding remarks
This study aimed to understand the cellular and molecular basis of the unique X-linked inheritance
pattern associated with PCDH19-GCE.
Our data highlights the ability of PCDH19 to cooperate with other NC PCDHs and dictate adhesion
specificities. In vivo, the generation of differential adhesion codes caused by mosaic expression of
PCDH19 leads to abnormal cell sorting, such that PCDH19-positive and PCDH19-null neuronal
progenitors segregate within the developing cortex. Furthermore, abnormal cell sorting is not present
in animals completely lacking PCDH19. Our findings suggest that disruption of cellular adhesion
codes is associated with the unique X-linked inheritance of PCDH19-GCE and that just a single
difference in PCDH expression is enough to disrupt the complex adhesion codes present in the
developing mammalian cortex.
Additional Methods
147
Additional Methods
Immunofluorescence
Immunostaining was performed as described in chapter 3. Additional antibodies used
goat anti-SOX3 1:300 (R&D Systems, AF2569 and guinea pig anti-CALBINDIN 1:400
(Synaptic Systems,214004). Images were acquired on a Leica SP5 Confocal microscope.
Modelling Size of Lattice-Like Adhesion Complexes
Modelling was performed according to experimental procedures outlined in (Rubinstein et
al., 2015).
References
148
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